Isoprenoid-dependent ras anchorage (idra) proteins

ABSTRACT

Disclosed is the identity of various Ras cell membrane anchor proteins. Also disclosed are methods for identifying other anchor proteins that bind isoforms of Ras, methods of identifying drug candidates that inhibit aberrant Ras activity and methods of determining therapeutic dosages of the drugs. Further disclosed are methods for disrupting aberrant Ras activity in vivo.

TECHNICAL FIELD

[0001] The present invention relates to Ras proteins, and morespecifically to interactions between Ras and other cellular proteins.

BACKGROUND OF THE INVENTION

[0002] Ras proteins must be anchored to the inner surface of the cellmembrane to function as cellular regulators of the signal transductionpathways controlling cell growth, differentiation, survival, andtransformation [Kloog et al., 1999]. Membrane anchorage of Ras proteinsis promoted by their C-terminal S-farnesylcysteine, by a stretch oflysines in K-Ras 4B, or by the S-farnesylcysteine and S-palmitoylmoieties in H— and N-Ras, suggesting the concept of a two-signalmechanism for Ras membrane targeting and association [Casey et al.,1989; Hancock et al., 1989; Cox et al, 1992]. In addition, intactsequences around the palmitoylation site are also required for propertargeting, indicating a three-signal mechanism [Willumsen et al., 1996].

[0003] The anchoring moieties of Ras proteins appear to target them tothe plasma membrane [Cox and Der, 1997] and possibly to specificmicrodomains [Song et al., 1996; Mineo et al., 1996; Engelman et al.,1997; Mineo et al., 1997]. The mechanism of the farnesyl-dependent Rasmembrane anchorage remains unknown. However, several experiments suggestthat the farnesyl moiety common to all Ras proteins serves as alipophilic lipid anchor and, in addition, confers functional specificityon Ras. For example, H-Ras modified by an inappropriate isoprenoid(e.g., by the C₂₀ geranylgeranyl group) has transforming activity butnot normal Ras function [Buss et al., 1989]. Other experiments showedthat modification of inactive unfarnesylated normal Ras by the fattyacid myristate results in activation of transforming activity, thussuggesting that myristoylated Ras (myr-Ras) cannot control normal Rasfunctions [Buss et al., 1989]. Furthermore, measurements of theassociation constants for Ras model peptides, modified by variouslipids, to lipid vesicles showed that farnesylated peptides bind with arelatively low affinity [Shahinian and Silvius, 1995; Schroeder et al.,1997]. These studies suggest that the branched side-chain structure offarnesyl is an inferior lipid glue when compared to other lipids such asmyristate or palmitate [Gelb et al., 1998]. Several lines of evidenceare consistent with the notions that Ras is not glued nonspecifically tothe cell membrane but rather is selectively tethered in specificmembrane microdomains, possibly associated with specific receptors oranchors [Siddiqui et al., 1998], and that interactions of Ras with suchdomains are dynamic in nature [Niv et al., 1999].

[0004] The experiments reviewed above raise the possibility that thefarnesyl group, common to all Ras proteins, acts as part of arecognition unit for specific anchorage lipids or protein(s) Ras in thecell membrane [Cox and Der, 1997]. On the assumption that Ras functionswould be inhibited by competitive displacement of the mature proteinfrom its putative membrane-anchorage domains, a series of organiccompounds resembling the farnesylcysteine of Ras proteins were designed[Marciano et al., 1995; Marciano et al., 1997; Aharonson et al., 1998].Among these compounds, S-trans, trans-farnesylthiosalicylic acid (FTS),was found to be a potent growth inhibitor of H-Ras-transformed Rat-I(EJ) fibroblasts. This compound and several of its active analogs wereeffective in a concentration range of 5-50

M and affected specifically the membrane-bound H-Ras protein in thesecells [Marciano et al., 1995; Marom et al., 1995; Aharonson et al.,1998; Haklai et al., 1998]. The observed stringent structuralrequirement for anti-Ras activity among S-prenyl analogs suggestedspecific protein binding [Aharonson et al., 1998]. Significantly, FTSand its C₂₀ geranylgeranyl analogue (GGTS), but not its Clo geranylanalogue (GTS) or its carboxy methylester analogue, inhibited growth ofRas-transformed cells [Aharonson et al., 1998]. The demonstration thatFTS inhibits the growth of fibroblasts transformed by ErbB2 actingupstream of Ras, but not the growth of cells transformed by v-Raf, whichunlike Raf-1 acts independently of Ras, suggested specificity of FTStowards Ras [Marom et al., 1995]. Mechanism of action studies showedthat FTS did not inhibit farnesylation of H-Ras [Marom et al., 1995]. Itaffected H-Ras membrane interactions in intact cells in vitro bydislodging the protein from its anchorage domains, which facilitated itsdegradation and thus reduced the total amount of cellular Ras [Haklai etal., 1998]. Although FTS and other S-prenyl analogues inhibit Rasmethylation in vitro [Marciano et al., 1995; Aharonson et al., 1998],its growth-inhibiting effects in intact cells occur at concentrationslower than those required for inhibition of methylation [Marom et al.,1995]. Additional studies showed that FTS inhibits growth of cellstransformed with the farnesylated but unmethylated K-Ras 4B (12V) CVYLisoform, confirming that methylation of Ras is not necessary fortransformation [Elad, et al., 1999]. Further studies demonstrated thatFTS also dislodges the normally processed K-Ras 4B (12V), N-Ras (13V)and N-Ras (61L) isoforms from membranes of rodent fibroblasts [Elad, etal. 1999; Jansen et al., 1999] and from membranes of human tumor celllines [Jansen et al., 1999; Weisz et al., 1999; Egozi et al., 1999]. Theeffects of FTS appeared to be specific to the Ras protein. For example,FTS did not dislodge the prenylated Gβγ-subunits of heterotriimericG-proteins from Rat-1 cell membranes and had no effect on myr-Ras inmyr-Ras-transformed cells [Haklai et al., 1998]. Recent studies alsoshowed that FTS did not reduce the amounts of prenylated Rac-1 and Rho Ain human melanoma cells [Jansen et al., 1999].

SUMMARY OF THE INVENTION

[0005] Applicants have isolated Ras-interacting proteins termed IDRA(isoprenoid-dependent Ras anchorage proteins). Ras-IDRA proteincomplexes were identified in extracts of membranes from H-Ras(12V)-transformed Rat-1 (EJ) cells. IDRAs were isolated from suchcomplexes and identified by MS and by specific antibodies as galectin-1,a mammalian protein associated with cell growth and transformation[Perillo et al., 1998]. On the basis of in vivo and in vitro studies,Applicants have established that this is an anchor protein for Ras, andparticularly the H-Ras isoform. In similar experiments, galectin-3 wasidentified as an anchor protein for the K-Ras isoform; galectin-7 andgalectin-8 were identified as anchor proteins for multiple Ras isoforms.

[0006] One aspect of the present invention is directed to a method foridentifying a cell membrane anchor protein that binds an isoform of Ras.The method entails preparing two reaction mixtures containing a sourceof a Ras protein and its anchor protein, e.g., intact cells, celllysate, cell membranes or fractions thereof. One reaction mixture alsocontains a Ras antagonist. A cross-linking agent is added to bothreaction mixtures whereby cross-linked complexes between Ras and cellmembrane proteins are produced. The cross-linked complexes formed inboth reaction mixtures are separated individually. The Ras-proteincomplex (formed in the reaction mixture without the antagonist) that isdisrupted by the Ras antagonist (present in the other reaction mixture)is identified. That complex is isolated from the other complexes, andthen the Ras protein is separated from the other protein(s) in thatcomplex. Preferred Ras antagonists are FTS and analogs thereof. In otherpreferred embodiments, the individual separation of the cross-linkedcomplexes and identification of the complex disrupted by antagonist areconducted by fractionating the two reaction mixtures side-by-side on agel. The method may be used to identify anchor proteins for prenylatedisoforms of Ras such H-Ras, K-Ras4A, K-Ras4B and N-Ras, generallyregarded as the “classic” Ras isoforms, as well as for prenylated Rasregulatory proteins such as Rac and Rho, and non-prenylated Rasregulatory proteins such as Rit and Rin. For purposes of the presentinvention, all such Ras proteins are referred to interchangeably as Rasisoforms or Ras proteins.

[0007] Another aspect of the present invention is directed to a methodfor identifying drug candidates that inhibit aberrant Ras activity. Thismethod entails preparing living cells or a reaction mixture containing aRas protein, one or more anchor proteins that bind the Ras protein andthe drug candidate, and determining the effect of the drug candidate onthe interaction of Ras and the anchor protein. The effect of the drug onRas-anchor interaction can be measured in a variety of ways. In oneembodiment, the change in the extent of dimerization of the Ras isoformis measured. In another embodiment, the change in the extent of bindingor activation of Raf protein is determined. In yet another embodiment,the change in the extent of binding between the Ras isoform and theanchor protein is measured in a reaction mixture or an intact cell. Inpreferred embodiments, the Ras isoform or the anchor protein isimmobilized on a matrix. In other preferred embodiments, the anchorprotein is galectin-1, galectin-3, galectin-7 or galectin-8.

[0008] Further aspects of the present invention are directed to a methodfor disrupting aberrant Ras activity in vivo, and compositions for usetherewith. The method entails administering to a patient exhibitingaberrant Ras activity specific oligonucleotides that bind and inactivatethe mRNA of an anchor protein for Ras. The specific oligonucleotidesbind mRNA of the anchor protein and thus decrease its expression that inturn decreases Ras activity. In preferred embodiments, the specificoligonucleotides bind galectin-1, galectin-3, galectin-7 or galectin-8mRNA. Combinations of antisense oligonucleotides that bind different ofthese proteins are also contemplated. The compositions contain anoligonucleotide that specifically targets a nucleic acid encoding a Rasanchor protein which binds (e.g., hybridizes) the nucleic acid andcauses degradation of the nucleic acid. Preferred antisense oligos bindnucleic acids encoding galectin-1, galectin-3, galectin-7 or galectin-8.

[0009] Yet another aspect of the present invention is directed to amethod of determining efficacious dosages of a Ras antagonist thatdisrupts Ras-anchor protein binding, comprising:

[0010] (i) contacting cells with the antagonist in vivo or in vitro;

[0011] (ii) collecting the cells following said contacting;

[0012] (iii) isolating cell membranes from the collected cells;

[0013] (iv) measuring decrease in anchor protein concentration per unitof cell membrane protein; and

[0014] (e) correlating the decrease with dosage of the Ras antagonist.

BRIEF DESCRIPTION OF THE DRAWINGS

[0015]FIG. 1 is a photograph of an electrophoretic gel illustrating useof Ras antibodies and chemical cross-linkers for identification of RasIDRA complexes that are sensitive to the Ras inhibitor, FTS. Rasantibodies (Ab) identify Ras and Ras-IDRA complex in EJ cell membranes.Membranes corresponding to 10⁶ control or FTS (50 μM)-treated EJ cellswere incubated in 50 mM sodium bicarbonate buffer, pH 8.5, containingprotease inhibitors and 2% DMSO (no cross-linker controls) or theindicated concentrations of the cross-linkers DSS or DSP. Samples ofTriton X-100 extracts of the membranes were subjected to SDS-PAGE undernon-reducing conditions followed by Western immunoblotting with Ras Ab.Open arrow corresponds to Ras (21 kDa) and closed arrow corresponds tothe major Ras-putative IDRA band (34-43 kDa) detected in the blot underthese conditions. This band is not detected in the blots of the noncross-linked samples or in the blots of the cross-linked samples ofFTS-treated cells.

[0016]FIG. 2 is a photograph of a Western blot of galectin-1 isolatedfrom cells with (+) and without (−) FTS treatment. FTS blocks theinteraction of Ras with its anchor, and reduces the amounts of anchor intreated cells.

[0017]FIG. 3 is a photograph of a Western blot of galectin-1 isolatedfrom cells in the presence of a control, FTS and GTS. FTS reduced theamount of membrane-associated galectin-1 by 90% while the inactiveanalog of FTS (GTS) had no effect.

[0018]FIG. 4 contains photographs of electrophoretic gels. Left Panel:membranes from five cell types, each with a different Ras isoform, werecross-linked, extracted, and fractionated on gels as in FIG. 1 (EJ andRat1 cells that contain oncogenic and wild-type H-Ras respectively;myr-Ras does not bind to any anchor protein). The 34-43 kDa band wasidentified with Ras antibody, extracted, and run on a second gel. RightPanel: The Ras proteins, galectin-1 (Gal-1) and galectin-3 (Gal-3),released under reducing conditions from the 34-43 kDa cross-linkedproteins form membranes isolated from cells transformed with variousoncogenic Ras isoforms.

[0019]FIG. 5 is a photograph of a Western blot, illustrating thatantisense Gal-1 reduces the expression of H-Ras (12V). C7-7 or 293Tcells transfected with oncogenic H-Ras in the vector pcDNA3 resulted inexpression of H-Ras protein (lane 3). When H-Ras was transfected withantisense to galectin-1, there was a decrease in Ras protein (lane 4),which was associated with a decrease in galectin-1 protein. The vectorand the antisense controls did not result in the production of Rasprotein (lanes 1 and 2).

[0020]FIG. 6 contains photographs of cells that produce H-Ras taggedwith green fluorescent protein (GFP). This allows for localization ofRas in live cells using fluorescent microscopy. Top: Followingtransfection with GFP-H-Ras (12V)+pcDNA3, only the membrane oftransformed cells was labeled with GFP tagged H-Ras as expected fromprevious studies [Niv et al., 1999]. Bottom: When GFP-H-Ras (12V) wastransfected with galectin-1 antisense, a large fraction of theGFP-labeled Ras was displaced into the cytoplasm from the cell membrane,as the galectin-1 anchor protein was reduced.

BEST MODE OF CARRYING OUT THE INVENTION

[0021] One aspect of the present invention is directed to a method foridentifying anchor proteins for the four farnesylated isoforms of Ras,namely H-Ras, K-Ras 4A, K-Ras 4B and N-Ras, the mutated forms of whichare known to be oncogenic. The Ras antagonist FTS and its active analogsare drugs containing the prenyl group farnesyl that specificallydisplaces activated Ras and oncogenic Ras from their binding sites onthe cell membranes. These prenylated drugs are sufficient to displaceand inactivate Ras even though Ras membrane binding is in partdetermined by the C-terminal amino acids of the Ras protein. The presentinvention utilizes this property to identify and isolate specificbinding sites or anchor proteins for farnesylated Ras.

[0022] Proteins such as Ras and its anchors are closely associated incell membranes and can be chemically joined together by a cross-linkingagent such as disuccininimidyl subarate (DSS) and dithiobis(succinirnidyl proprionate) (DSP). The crossed linked Ras/anchorprotein(s) complexes are larger and will migrate slowly on SDS gelscompared to Ras. Candidate molecules for isolation are complexes thatstain with anti-Ras antibodies, that are larger than Ras, and that arepresent in markedly reduced concentrations in membranes that arepretreated with the Ras antagonists. Once the cross-linked complex ispurified, the candidate anchor protein for the farnesylated Ras isreleased from Ras by reversing the chemical cross-linking and isolatedsuch as by fractionation on a SDS gel. Separation of the Ras proteinfrom the putative anchor is conducted in accordance with standardtechniques such as liquid chromatography and gel electrophoresis. Thereleased anchor protein may then be extracted from the gel andsequenced. This approach may also be used to identify the anchors forall other isoforms of Ras.

[0023] In other embodiments, anchor proteins for isoforms of Ras thatare prenylated with groups other than farnesyl as well as those that arenot prenylated are identified. FTS and its active prenylated analogsalso competitively displace regulatory proteins that are anchored to thecell membrane by a prenyl group other than farnesyl such as geranylgeranyl. Suitable analogs of FTS include 5-fluoro-FTS, 5-chloro-FTS,4-chloro-FTS, 2-chloro-5-farnesylaminobenzoic acid,3-farnesylthio-cis-acrylic acid, farnesyl thionicoatinic acid, orS-farnesyl-methylthiosalicylic acid. These prenylated drugs togetherwith cross-linking reagents permit isolation and identification ofanchor proteins for prenylated proteins such as Rac and Rho. Other Rasassociated regulatory proteins such as Rit, Rin and many nonprenylatedisoforms of Ras are bound to cell membranes without the aid of a prenylgroup. The interaction of these latter regulatory proteins with the cellmembrane is dependent upon a small portion of their amino acidsequences. Nonetheless, small organic molecules that interact with theseamino acid sequences can completely displace these proteins from theirmembrane anchor sites. Thus, the anchor sites are identified using thecombination of the displacing drug and cross-linking reagents. Suitableorganic molecules can be identified from large chemical libraries.

[0024] These methods, as well as other methods disclosed herein, may beconducted using whole cells, cell lysate or homogenate, or isolated cellmembranes or fragments thereof. Preferred whole cells include NIHfibroblasts transformed with oncogenic K-Ras 4B (12V), H-Ras (12V) orN-Ras (13V), 518A2/N-Ras melanoma cells, 607B melanoma cells, Panc-Icells containing oncogenic K-Ras, transformed Rat-1 EJ cells andMC-MA-11 cells.

[0025] Another aspect of the present invention is directed to methodsfor screening chemicals for identification of drugs that block theinteractions of Ras isoforms or proteins with their cognate anchorproteins. The methods identify molecules that displace a regulatoryprotein such as Ras from any cellular anchorage site. The functions ofsuch anchorage proteins are to allow the regulatory proteins such as Rasto interact with cellular membranes so that they can dimerize andcombine with cytosplasmic factors to enhance or propagate theiractivities. Thus, the anchor protein may be obtained from any cellularcompartment. In general, the methods involve the competitive inhibitionof the interaction of two proteins (e.g., the Ras isoform and anchorprotein) by the drug candidate. As a result, any competitive bindingassay that involves interaction of three or four components may beemployed. Many of these assays have been developed to measure hormonesin biological fluids, hormone receptor interactions, andantibody/antigen interactions and interaction of regulatory proteinswith activators and suppressors. Such binding reactions are usually madeat equilibrium or in real time depending on the instrurnentation. Ineach instance, the endpoint of the assay directly or indirectly measuresthe interaction of the drug with one or both proteins or quantifies thebiological consequences of this interaction. Depending upon theparticular method employed, the anchor protein and/or the Ras protein isimmobilized on a matrix or is in solution. In addition, either or bothproteins may be detectably labeled e.g., with a fluorescent protein suchas green fluorescent protein (GFP) or yellow fluorescent protein, suchas when movement of the protein(s) from one location within the cell toanother is being observed.

[0026] In one embodiment, the effect of the drug candidate on theinteraction between the Ras protein and the anchor protein is determinedby measuring the extent to which dimer formation of Ras protein isreduced. For Ras and its isoforms to be active, they must be recruitedto the cell membrane where they form dimers in association with theiranchors. The Ras dimer then interacts with and activates Raf protein andother cytoplasmic factor(s). This complex then initiates a regulatorycascade. The drug disruption of dimer formation or the recruitment ofother molecules such as Raf is quantified. Methods for quantification ofRas dimer formation and Raf activity e.g., by determining binding of Rafto Ras, are described in Inouye, et al., (2000). In another embodiment,the effect of the drug candidate on the interaction between the Rasprotein and the anchor protein is determined by measuring the extent towhich cross-linking of the Ras protein with the anchor protein isreduced. Samples of Ras/anchor complex are reacted with and without thedrug candidate followed by treatment with a cross linking agent. Theamount of complexation with and without the drug is measured.

[0027] In yet another embodiment, the effect on the interaction isdetermined by measuring the extent of Ras binding with the anchorprotein. The method may be conducted by observing movement of protein ina living cell. Further, the method may be conducted with both proteinsin solution or wherein one of the proteins may be immobilized on amatrix such as a column. The proteins are identified in accordance withstandard techniques, such as by an antibody, a fluorescent tag, or byprotein-protein interaction. Examples of protein interactions include:(i) an affinity column or membrane with one protein coupled to thematrix and the drug prevents binding of the second protein; (ii) surfaceplasmon resonance e.g., as measured with “BIAcore” biosensorinstrumentation where one protein in solution interacts with the otherprotein anchored to the cell of the Biocor, wherein protein/proteininteractions and the ability of drugs to disrupt such interactions aremeasured in real time (an advantage of this technology being thataffinity constants can be measured); (iii) color transfer generated byinteraction of two tagged proteins in solution and how this isinfluenced by drugs is also measured in real time; (iv) interaction of afluorescent tagged protein with an untagged protein which is coated onthe surface of an object such as a sheep red blood cells calowsmeasurement of drug induced interactions with a FACScan machine (GuavaPersonal Cytometer); and (v) interaction of a tagged protein in amicrotiter plate which allows drug modulation of protein/proteininteraction at equilibrium using a microtiter plate reader.

[0028] In preferred embodiments, the method is conducted usinggalectin-1, galectin-3, galectin-7 or galectin-8. Galectin-1 is aprotein known to bind beta-galactoside. The amino acid sequence of therat protein is as follows:MACGLVASNLNLKPGECLKVRGELAPDAKSFVLNLGKDSNNLCLHFNPRFNAHGDANTIVCNSKDDGTWGTEQRETAFPFQPGSITEVCITFDQADLTIKLPDGHEFKFPNRLNMEAINYMAADGDFKIKCVAFE (SEQ ID NO:1). See, Clerch, et al. (1988).The corresponding nucleotide sequence is (SEQ ID NO:2)5′ATGGCCTGTGGTCTGGTCGCCAGCAACCTGAATCTCAAACCTGGGGAATGTCTCAAAGTTCGGGGAGAGCTGGCCCCGGACGCCAAGAGCTTTGTGTTGAACCTGGGGAAAGACAGCAACAACCTGTGCCTACACTTCAACCCCCGCTTCAACGCCCACGGAGATGCCAACACCATTGTGTGTAACAGCAAGGACGATGGGACCTGGGGAACAGAACAACGGGAGACTGCCTTCCCTTTCCAGCCTGGGAGCATCACGGAGGTGTGCATCACCTTTGACCAGGCTGACCTGACCATCAAGCTGCCAGACGGGCATGAATTCAAATTCCCCAACCGCCTCAACATGGAGGCCATCAACTACATGGCGGCGGATGGTGACTTCAAGATTAAGTGTGTGGC CTTTGAGTGA 3′

[0029] Galectin-1 obtained from mouse and human cells has also beenreported. See, Wilson, et al. (1989) and Gitt, et al. (1991). The aminoacid and corresponding nucleotide sequences of human galectin-1 are setforth as SEQ ID NOS: 3 and 4 respectively. (SEQ ID NO:3)MACGLVASNLNLKPGECLRVRGEVAPDAKSFVLNLGKDSNNLCLHFNPRFNAHGDANTIVCNSKDGGAWGTEQREAVFPFQPGSVAEVCITFDQALNLTV KLPDGYEFKFPNRINLEATNYMAADGDFKIKCVAFD

[0030] Coding sequence: (SEQ ID NO:4)5′ ATGGCTTGTGGTCTGGTCGCCAGCAACCTGAATCTCAAACCTGGAGAGTGCCTTCGAGTGCGAGGCGAGGTGGCTCCTGACGCTAAGAGCTTCGTGCTGAACCTGGGCAAAGACAGCAACAACCTGTGCCTGCACTTCAACCCTCGCTTCAACGCCCACGGCGACGCCAACACCATCGTGTGCAACAGCAAGGACGGCGGGGCCTGGGGGACCGAGCAGCGGGAGGCTGTCTTTCCCTTCCAGCCTGGAAGTGTTGCAGAGGTGTGCATCACCTTCGACCAGGCCAACCTGACCGTCAAGCTGCCAGATGGATACGAATTCAAGTTCCCCAACCGCCTCAACCTGGAGGCCATCAACTACATGGCAGCTGACGGTGACTTCAAGATCAAATGTGTGG CCTTTGACTGA 3′

[0031] Galectin-1 DNAs from the mouse and rat are not identical but theypossess 94% sequence similarity. The corresponding amino acid sequencespossess 95% sequence similarity. The amino acid sequence of the mousegalectin-1 is as follows: (SEQ ID NO:5)MACGLVASNLNLKPGECLKVRGEVASDAKSFVLNLGKDSNNLCLHFNPPYNAHGDANTIVCNTKEDGTWGTEHREPAFPFQPGSITEVCITFDQADLTIKLPDGHEFKFPNRLNMEATNYMAADGDFKIKCVAFE

[0032] The molecular weight of galectin-3 varies within and amongspecies and ranges from 29-34 kD. See, Liu, et al. (1987) (rat); Pillai,(1990) (human); and Cherayil, et al. (1989) (mouse). The amino acid andcorresponding nucleic acid sequences for two human galectin-3 proteinsare set forth below. MADNFSLHDALSGSGNPNPQGWPGAWGNQPAGAGGYPGASYPGAYPGQAPPGAYPGQAPP (SEQ ID NO:6) GAYPGAPGAYPGAPAP GVYPGPPSGPGAYPSSGQPSATGAYPATGPYGAPAGPLTVPYNLPL PGG VVPRMLITILGTVKPNA NRIALDFQRGNDVAFHFNPRFNENNRRVIVCNTKLDNNWGR BERQ SVFPFESGK PFKIQVLVEPDHFKVAVNDAHLLQYNHRVKKLNEISKLGISGDIDLTS ASYTMI Coding sequence: 5′ ATGGCAGACAATTTTTCGCTCCATGAT GCGTTATCTG GGTCTGGAAA CCCAAACCCT (SEQ ID NO:7)CAAGGATGGCCTGGCGCATG GGGGAACCAG CCTGCTGGGG CAGGGGGCTA CCCAGGGGCTTCCTATCCTGGGGCCTACCC CGGGCAGGCA CCCCCAGGGG CTTATCCTGG ACAGGCACCTCCAGGCGCCTACCCTGGAGC ACCTGGAGCT TATCCCGGAG CACCTGCACC TGGAGTCTACCCAGGGCCACCCAGCGGCCC TGGGGCCTAC CCATCTTCTG GACAGCCAAG TGCCACGGGAGCCTACCCTGCCACTGGCCC CTATGGCGCC CCTGCTGGGC CACTGATTGT GCCTTATAACCTGCCTTTGCCTGGGGGAGT GGTGCCTCGC ATGCTGATAA CAATTCTGGG CACGGTGAAGCCCAATGCAAACAGAATTGC TTTAGATTTC CAAAGAGGGA ATGATGTTGC CTTCCACTTTAACCCACGCTTCAATGAGAA CAACAGGAGA GTCATTGTTT GCAATACAAA GCTGGATAATAACTGGGGAAGGGAAGAAAG ACAGTCGGTT TTCCCATTTG AAAGTGGGAA ACCATTCAAAATACAAGTACTGGTTGAACC TGACCACTTC AAGGTTGCAG TGAATGATGC TCACTTGTTGCAGTACAATCATCGGGTTAA AAAACTCAAT GAAATCAGCA AACTGGGAAT TTCTGGTGACATAGACCTCACCAGTGCTTC ATATACCATG ATATAA 3′ B001120. Homo sapiens, lec . .. [gi:12654570]MADNFSLHDALSGSGNPNPQGWPGAWGNQPAGAGGYPGASYPGAYPGQAPPGAYPGQAPPGAYP (SEQ IDNO:8) GAPGAYPGAPGVYPGPPSGPGAYPSSGQPSATGAYPATGPYGAPAGPLIVPYNLPLPG GVVPRMLITILGTV KPNAINRLALDFQRGNDVAFKFNPRFNENNRRVIVCNTKLDNNWGREERQSVFPFESGKPFKIQVLVEPDHFKVAVNDAHLLQYNHRVKKLNEISKLGISGDIDLTSASYTMI Codingsequence: 5′ ATGGCAG ACAATTTTTC GCTCCATGATGCGTTATCTG GGTCTGGAAACCCAAACCCT (SEQ ID NO:9) CAAGGATGGC CTGGCGCATG GGGGAACCAGCCTGCTGGGGDAGGGGGCTA CCCAGGGGCT TCCTATCCTG GGGCCTACCC CGGGCAGGCACCCCCAGGGGCTTATCCTGG ACAGGCACCT CCAGGCGCCT ACCCTGGAGC ACCTGGAGCTTATCCCGGAGCACCTGCACC TGGAGTCTAC CCAGGGCCAC CCAGCGGCCC TGGGGCCTACCCATCTTCTGGACAGCCAAG TGCCACCGGA GCCTACCCTG CCACTGGCCC CTATGGCGCCCCTGCTGGGCCACTGATTGT GCCTTATAAC CTGCCTTTGC CTGGGGGAGT GGTGCCTCGCATGCTGATAACAATTCTGGG CACGGTGAAG CCCAATGCAA ACAGAATTGC TTTAGATTTCCAAAGAGGGAATGATGTTGC CTTCCACTTT AACCCACGCT TCAATGAGAA CAACAGGAGAGTCATTGTTTGCAATACAAA GCTGGATAAT AACTGGGGAA GGGAAGAAAG ACAGTCGGTTTTCCCATTTGAAAGTGGGAA ACCATTCAAA ATACAAGTAC TGGTTGAACC TGACCACTTCAAGGTTGCAGTGAATGATGC TCACTTGTTG CAGTACAATC ATCGGGTTAA AAAACTCAATGAAATCAGCAAACTGGGAAT TTCTGGTGAC ATAGACCTCA CCAGTGCTTC ATATACCATGATATAA 3′

[0033] Three additional human galectin-3 sequences, as well as a rat andmouse galectin-3 sequence, are set forth below. M35368 (human)MADNFSLHDALSGSGNPNPQGWPGAWGNQPAGAGGYPGASYPGA (SEQ ID NO:10)YPGQAPPGAYPGQAPPGAYPGALPGAYPGAYAPGVYPGPPSGPGAYPSSGQPSAPGAYPATGPYGAPAGPLIVPYNLPLPGGVVPRMLITILGTVKPN ANRIALDFQRGNDVAFHFNPRFNENNRRVIVCNTKIDNNW GREERQSVFPFESGKPFKIQVLVEPDHFKVAVNDAIHLLQYNIIRVKKLNEISKLGISGDIDLTSASYTMI NM_002306 (human)MADNFSLHDALSGSGNPNPQGWPGAWGNQPAGAGGYPGASYPGA (SEQ ID NO:11)YPGQAPPGAYPGQAPPGAYHGAPGAYPGAPAPGVYPGPPSGPGAYPSSGQPSAPGAYPATGPYGAPAGPLTVPYNLPLPGGVVPRMLITILGTVKPNANRIALDFQRGNDVAFHFNPRFNENNRRVIVCNTKLDNNWGREERQSVFPFESGKYFKIQVLVEPDHFKVAVNDAHLLQYNHRVKIKLNEISKLGISGDIDLTSASYTMI S59012 (human)MADNFSLHDALSGSGNPNPQGWPGAWGNQPAGAGGYPGASYPGA (SEQ ID NO:12)YPGQAPPGAYPGQAPPGAYPGAPGAYPGAPAPGVYPGPPSGPGAYPSSGQPSATGAYPATGPYGAPAGPLIVPYNLPLPGGVVPRMLITILGTVKPNANRIALDFQRGNDVAFHFNPRFNENNRRVIVCNTKLDNNWGREERQSVFPFESGKPFKIQVLVEPDPYKVAVNDAHLLQYNHRVKKLNEISKLGISGDIDLTSASYTMI P08699 (rat) MADGFSLNDA LAGSGNPNPQGWPGAWGNQP GAGGYPGASY PGAYPGQAPP (SEQ ID NO:13) GGYPGQAPPS AYPGPTGPSAYPGPTAPGAY PGPTAPGAFP GQPGGPGAYP SAPGAYPSAP GAYPATGPFG APTGPLTVPYDMPLPGGVMP RMLITIIGTV KPNANSITLN FKKGNDIAFH FNPRFNENNR RVIVCNTKQDNNWGREERQS AFPFESGKPF KIQVLVEADH FKVAVNDVHL LQYNHRMKNL REISQLGIIGDITLTSASHA MI P16110 (mouse) MADSFSLNDA LAGSGNPNPQ GYPGAWGNQP GAGGYPGAAYPGAYPGQAPP (SEQ ID NO:14) GAYPGQAPPG AYPGQAPPSA YPGPTAPGAY PGPTAPGAYPGQPAPGAFPG QPGAPGAYPQ CSGGYPAAGP GVPAGPLTV PYDLPLPGGV MPRMLITIMGTVKPNANRIV LDFRRGNDVA FHFNPRFNEN NRRVIVCNTK QDNNWGKEER QSAFPFESGKPFKIQVLVEA DHFKVAVNDA HLLQYNHRMK NLREISQLGI SGDITLTSAN HAMI

[0034] In other embodiments, the method is conducted using galectin-7and/or galectin-8, which Applicants have also found to function as cellmembrane anchors for Ras isoforms. Amino acid and corresponding nucleicacid sequences of human galectin-7 and -8 are set forth below.

[0035] Galectin-7 L07769. Homo sapiens galectin-7 [gi:182131]MSNVPHKSSLPEGTRPGTVLRIRGLVPPNASRFHVNLLCGEEQG (SEQ ID NO:15)SDAALHFNPRLDTSEVVFNSKEQGSWGREERGPGVPFQRGQPFEVLIIASDDGFKAVVGDAQYHHFRHRLPLARVRLVEVGGDVQLDSVRIF Coding sequence: 5′ATGTCCAACGTCCCCCACAAGT CCTCGCTGCC CGAGGGCATCCGCCCTGGCA CGGTGCTGAG (SEQ ID NO:16)AATTCGCGGC TTGGTTCCTC CCAATGCCAG CAGGTTCCATGTAAACCTGC TGTGCGGGGAGGAGCAGGGC TCCGATGCCG CCCTGCATTT CAACCCCCGGCTGGACACGT CGGAGGTGGTCTTCAACAGC AAGGAGCAAG GCTCCTGGGG CCGCGAGGAGCGCGGGCCGG GCGTTCCTTTCCAGCGCGGG CAGCCCTTCG AGGTGCTCAT CATCGCGTCAGACGACGGCT TCAAGGCCGTGGTTGGGGAC GCCCAGTACC ACCACTTCCG CCACCGCCTGCCGCTGGCGC GCGTGCGCCTGGTGGAGGTG GGCGGGGACG TGCAGCTGGA CTCCGTGAGGATCTTCTGA 3′ Galectin-8AY037304. Homo sapiens beta . . . [gi:14626473]MMLSLNNLQNIIYSPVIPYVGTIPDQLDPGTLTVICGHVPSDAD (SEQ ID NO:17)RFQVDLQNGSSVKPRDVAYHFNPRFKPAGCTVCNTLTNEKWGBEEITYDTPFK REKSFEIVIMVLKDKFQVPKSGTPQLPSNRGGDISKIAPRTVYTKSKD S TVNHTLTCTKIPPTNYVSKILPFAALNTPMGPGGTVVVKGEVNANAKSFNVDLLAGKSKHIALHLNPRLNIKAFVRNSFLQESWGEEEPNITSFPFSPGMYFEMIIYCDVREFKVAVNGVHSLEYKHR FKELSSIDTLEINGDIHLLEVRSW Codingsequence: 5′ATGATGTTGT CCTTAAACAA CCTACAGAAT ATCATCTATA GCCCGGTAATCCCGTATGTT GGCACCATTC CCGATCAGCT GGATCCTGGA ACTTTGATTG TGATATGTGGGCATGTTCCT AGTGACGCAG ACAGATTCCA GGTGGATCTG CAGAATGGCA GCAGTGTGAAACCTCGAGCC GATGTGGCCT TTCATTTCAA TCCTCGTTTC AAAAGGGCCG GCTGCATTGTTTGCAATACT TTGATAAATG AAAAATGGGG ACGGGAAGAG ATCACCTATG ACACGCCTTTCAAAAGAGAA AAGTCTTTTG AGATCGTGAT TATGGTGCTA AAGGACAAAT TCCAGGTTCCAAAGTCTGGC ACGCCCCAGC TTCCTAGTAA TAGAGGAGGA GACATTTCTA AAATCGCACCCAGAACTGTC TACACCAAGA GCAAAGATTC GACTGTCAAT CACACTTTGA CTTGCACCAAAATACCACCT ACGAACTATG TGTCGAAGAT CCTGCCATTC GCTGCAAGGT TGAACACCCCCATGGGCCCT GGCGGCACTG TCGTCGTTAA AGGAGAAGTG AATGCAAATG CCAAAAGCTTTAATGTTGAC CTACTAGCAG GAAAATCAAA GCATATTGCT CTACACTTGA ACCCACGCCTGAATATTAAA GCATTTGTAA GAAATTCTTT TCTTCAGGAG TCCTGGGGAG AAGAAGAGAGAAATATTACC TCTTTCCCAT TTAGTCCTGG GATGTACTTT GAGATGATAA TTTATTGTGATGTTAGAGAA TTCAAGGTTG CAGTAAATGG CGTACACAGC CTGGAGTACA AACACAGATTTAAAGAGCTC AGCAGTATTG ACACGCTGGA AATTAATGGA GACATCCACT TACTGGAAGTAAGGAGCTGG

[0036] TAG 3′ (SEQ ID NO:18). In the methods of the present invention,fragments of the anchor proteins that bind the Ras protein may also beused. Thus, the term “anchor protein” as used herein includes suchfragments as well as the full-length proteins.

[0037] Another aspect of the present invention is directed to a methodfor reducing or inhibiting aberrant Ras activity in vivo. In general,aberrant Ras activity is manifested by uncontrolled mitosis. Diseasescharacterized by this phenomenon include cancers and variousnon-malignancies such as autoimmune diseases (e.g., type 1 diabetes,lupus and multiple sclerosis), cirrhosis, graft rejection,atherosclerosis, polycystic kidneys and post-angioplasty restenosis.Preferred indications are diseases characterized by proliferation of thecells of the diseased organ, including a proliferation of T-cells. Themethod entails administering to patients oligonucleotides that are inthe antisense orientation to the mRNAs for galectin-1, galectin-3 and/oranother Ras anchor protein such as galectin-7 or galectin-8. They aredesigned based on sequences that show the most potent effects ontranslation of the protein and minimizing non-antisense effects.

[0038] Factors taken into consideration in the design of antisense DNAsinclude the length of an oligonucleotide, its binding affinity andaccessibility of the target RNA, resistance to degradation by endogenousnucleases, permeability through target cell membranes. Tens ofoligonucleotides may be screened on target cells in culture to selectthe most potent inhibitors (Wagner, et al., 1993). Tumor cells areparticularly preferred target cells. In general, most regions of the RNA(e.g., 5′- and 3′-untranslated, AUG initiation sites, splice junctionsand introns) may be targeted using antisense oligonucleotides. Enhancedbinding affinity and nuclease stability are critical for antisenseactivity. Optimal length of the oligonucleotides varies, but in general,is about 15 nucleotides. The sequence of an antisense compound does notbe 100% complementary to that of its target nucleic acid to bespecifically hybridizable. An antisense compound is specificallyhybridizable when binding of the compound to the target DNA or RNAmolecule interferes with the normal function of the target DNA or RNA tocause a loss of utility, and there is a sufficient degree ofcomplementarity to avoid non-specific binding of the antisense compoundto non-target sequences under conditions in which specific binding isdesired, i.e., under physiological conditions in the case of in vivoassays or therapeutic treatment, and in the case of in vitro assays,under conditions in which the assays are performed. The inclusion ofphosphorathioate-modified oligonucleotides that contain the C-5 propyneanalogs of uridine and cytidine improve binding and stability of theantisense oligos. (Wagner, 1993). Modest increases in activity can alsobe achieved by delivering the antisense oligos via a liposome. Forexample, up to a 10-fold increase in biological activity ofoligonucleotides in vitro is achieved by complexing with the serumresistant cationic liposome, GS2888. See also WO 96/40062 whichdiscloses methods for encapsulating high molecular weight nucleic acidsin liposomes; U.S. Pat. No. 5,264,221 which discloses protein-bondedliposomes and asserts that the contents of such liposomes may include anantisense RNA; U.S. Pat. No. 5,665,710 which describes certain methodsof encapsulating oligodeoxynucleotides in liposomes; WO 97/04787 whichdiscloses liposomes comprising antisense oligonucleotides targeted tothe raf gene.

[0039] In preferred embodiments, the oligonucleotides are formulated forhuman use by dissolution in a saline solution for IV administration. Adose response effect is expected at doses between 0.06 and 7 mg/kg/dayfor one to two weeks of continuous treatment (Wagner, 1995). Theantisense oligonucleotides bind the nucleic acid e.g., mRNA, of theanchor protein, thus causing degradation of the mRNA and secondarilycausing a decrease in the concentration of Ras. The antisense compoundscontaining the oligonucleotides are prepared in accordance with knownprocedures such as those referenced in U.S. Pat. No. 6,294,382.

[0040] Antisense mRNA that bind galectin-1 mRNA, for example, may bedesigned by testing sequences selected along the length of the antisensemRNA and testing them in vitro for potency before using them in vivo.The full-length antisense for human galectin-1 is set forth below.

[0041] TCAGTCAAAGGCCACACATTTGATCTTGAAGTCACCGTCAGCTGCCATGTAGTTGATGGCCTCCAGGTTGAGGCGGTTGGGGAACTTGAATTCGTATCCATCTGGCAGCTTGACGGTCAGGTTGGCCTGGTCGAAGGTGATGCACACCTCTGCAACACTTCCAGGCTGGAAGGGAAAGACAGCCTCCCGCTGCTCGGTCCCCCAGGCCCCGCCGTCCTTGCTGTTGCACACGATGGTGTTGGCGTCGCCGTGGGCGTTGAAGCGAGGGTTGAAGTGCAGGCACAGGTTGTTGCTGTCTTTGCCCAGGTTCAGCACGAAGCTCTTAGCGTCAGGAGCCACCTCGCCTCGCACTCGAAGGCACTCTCCAGGTTTGAGATTCAGGTTGCTGGCGACCAGACCACAAGCCAT (SEQ ID NO:19).Preferred galectin-1 antisense oligonucleotides are as follows:

[0042] AAGTCACCGTCAGCTGCCATGTAGT (SEQ ID NO:20);

[0043] GATGCACACCTCTGCAACACTTC (SEQ ID NO:21);

[0044] TCAGCACGAAGCTCTTAGCGTCAG (SEQ ID NO:22);

[0045] GCACTCGAAGGCACTCTCCAGG (SEQ ID NO:23); and

[0046] GGTTGCTGGCGACCAGACCACA (SEQ ID NO:24).

[0047] The full-length antisense for human galectin-3 is set forthbelow. TTATATCATGGTATATGAAGCACTGGTGAGGTCTATGTCACCAGAAATTCCCAGTTTGCTGATTTCATTGAGTTTTTTAACCCGATGATTGTACTGCAACAGTGAGCATCATTCACTGCAACCTTGAAGTGGTCAGGTTCAACCAGTACTTGTATTTTGAATGGTTTCCCACTTTCAAATGGGAAAACCGACTGTCTTTCTTC CCTTCCCCAGTTATTATCCAGCTTTGTATTGCAAACAATGACTCTCCTGTTGTTCTCATTGAAGCGTGGGTTAAAGTGGAAGGCAACATCATTCCCTCTTTGGAAATCTAAAGCAATTCTGTTTGCATTGGGCTTCACCGTGCCCAGAATTGTTATCAGCATGCGAGGCACCACTCCCCCAGGCAAAGGCAGGTTATAAGGCACAATCAGTGGCCCAGCAGGGGCGCCATAGGGGCCAGTGGCAGGGTAGGCTCCGGGGGCACTTGGCTGTCCAGAAGATGGGTAGGCCCCAGGGCCGCTGGGTGGCCCTGGGTAGACTCCAGGTGCGGTGCTCCGGGATAAGCTCCAGGTGCTCCATGGTAGGCGCCTGGAGGTGCCTGTCCAGGATAAGCCCCTGGGGGTGCCTGCCCGGGGTAGGCCCCAGGATAGGAAGCCCCTGGGTAGCCCCCTGCCCCAGCAGGCTGGTTCCCCCATGCGCCAGGCCATCCTTGAGGGTTTGGGTTTCCAGACCCAGATAACGCATCATGGAGCGAAAAATTGTCTGCCAT (SEQ ID NO:25). Preferred galectin-3antisense oligonucleotides are as follows:

[0048] TATATGAAGCACTGGTGAGGTC (SEQ ID NO:26);

[0049] GAAGCGTGGGTTAAAGTGGAAGGC (SEQ ID NO:27);

[0050] TTGTTATCAGCATGCGAGGCACCACTCCCC (SEQ ID NO:28);

[0051] CACTTGGCTGTCCAGAAGATG (SEQ ID NO:29);

[0052] GATAAGCTCCAGGTGCTCCATGGTAG (SEQ ID NO:30); and

[0053] TCCAGACCCAGATAACGCAT (SEQ ID NO:31).

[0054] Preferred antisense oligonucleotides that bind galectin-7 andgalectin-8 mRNA are as follows: Galectin-7 antisense oligo:

[0055] 5′ TGTGGGGGACGTTGGACAT 3′ (SEQ ID NO:32)

[0056] Galectin-8 antisense oligo:

[0057] 5′ TGTTTAAGGACAACATCAT 3′ (SEQ ID NO:33).

[0058] Yet another aspect of the present invention is directed to amethod of determining efficacious dosages of a Ras antagonist thatdisrupts Ras-anchor protein binding. In general, the method entailscontacting cells with the antagonist in vivo or in vitro, collecting thecells following the contacting, isolating cell membranes from thecollected cells, measuring decrease in anchor protein concentration perunit of cell membrane protein, and correlating the decrease with dosageof the Ras antagonist. In a preferred embodiment, a method for measuringthe biological action of FTS and its analogs in vivo and in vitro isbased on the suppression of the immunoassayable galectin-1 inH-Ras-transformed tumors. The basis of this assay depends upon the dosedependent loss of galectin-1 from cell membranes by FTS. Under maximalstimulation of tumor cells with FTS, 90% of galectin-1 is lost frommembrane. This allows for an excellent dose response. A variation ofthis assay uses drug induced suppression of the anchor proteins innormal human lymphocytes isolated from patients being treated with FTSin phase 1 clinical trials. A dose of FTS or any other Ras antagonistthat maximally suppresses galectin-1 (or the respective anchor proteinof the antagonist) should be a dose that produces effects on otherbiological endpoints in vivo. When such assay is in mice and humans,therapeutically efficacious doses of Ras antagonists for humans can bedetermined.

[0059] Various aspects of the present invention are further illustratedby the following examples. The presentation of these examples is by noway intended to limit Applicants' invention in any way. Unless otherwisespecified, all percentages are by weight.

EXAMPLE 1

[0060] We used chemical cross-linkers to isolate a protein whoseinteraction with Ras could be blocked by FTS. We identified such aprotein that forms FTS-sensitive complexes with H-Ras (12V) intransformed Rat-1 (EJ) cells. This protein was isolated from suchcomplexes and identified by mass spectrophotometry (MS) and by specificantibodies such as galectin-1, a mammalian galactose-binding proteinknown to be associated with cell growth and transformation. Cross-likingof H-Ras to galectin-1 detected in intact EJ cells and in cell membraneswas independent of galectin-1 sugar-binding activity. FTS (but not itsinactive analog, GTS) decreased the levels of endogenous galectin-1 inEJ cells in parallel with the decrease in Ras. Galectin-1 seems tointeract preferentially with farnesylated H-Ras (12V). K-Ras 4B (12V)did interact with galectin-1 though less efficiently than H-Ras.Galectin-3 interacted with K- and H-Ras. Activated N-Ras (13V) did notform complexes with galectin-1 or galectin-3. Co-expression ofgalectin-1 antisense RNA and H-Ras (12V) in two cell lines resulted in adecrease in Ras protein as detected by Western blots. Using confocalmicroscopy expression of galectin-1 antisense resulted in release ofH-Ras labeled with green fluorescent protein (GFP) from the membranes oflive cells. Thus, H-Ras (12V) and galectin-1 seem to interact in thecell membrane and to cooperate in cell transformation. These resultsprovide a link between Ras transformation and the knownsugar-independent mitogenic and transforming potentials of galectin-1,which, like those of activated Ras, are associated with many types ofhuman malignancies.

[0061] 1. Identification of Ras-Interacting Proteins Sensitive to theRas Inhibitor FTS

[0062] The somewhat limited, but fast, lateral mobility of Ras in thecell membrane suggests that interactions of Ras with other proteins arelikely to be dynamic and transient [Niv et al., 1999]. We used chemicalcross-linkers in an attempt to identify the rapidly dissociatingcomplexes of Ras and Ras-interacting proteins. The Ras inhibitor FTS,which was shown to relieve constraints on the lateral mobility of Ras[Niv et al., 1999], was used as an analytical tool in order to identifycomplexes that are sensitive to this inhibitor. Accordingly, theanalytical steps were performed with control and with FTS-treated EJcells in combination with the cross-linkers DSS and DSP, the latter ofwhich is reducible. When membranes of control and FTS-treated cells wereexposed to these cross-linkers solubilized and fractionated onSDS-containing gels, Ras-immunoreactive bands were clearly detected at34-43 kDa, 50 kDa, and 70 kDa (FIG. 1). These complexes were notdetected in the absence of the cross-linkers. The broadband at 34-43 kDawas not present in cells (data not shown) or cell membranes (See FIG. 1)after treatment with FTS. The proteins in this band fit the criterion ofRas proteins bound to their specific anchors (IDRA) because FTS and itsactive analogs prevent this binding. Interaction of Ras with the IDRAswas not disrupted with analogs of FTS that had no anti-Ras activity ontumor cells (data not shown). This experiment showed how to identifyproteins that interact with Ras and an anti-Ras drug.

[0063] 2. Purification of Ras-Interacting (Anchor) Proteins from EJCells.

[0064] Triton X-100 extracts of the membranes containing Ras complexesformed by cross-linking with DSP were used for subsequent purificationsteps. The first steps were performed in the absence of reducingreagents to enable the purification to be followed by Ras antibodies.The release of Ras from the putative IDRA proteins was performed only atthe final fractionation step. The details of the purification aresummarized by way of the following flow diagram.

[0065] Purify 34-43 kDa Ras-protein complexes.

[0066] Run concentrate Mono Q pool on SDS-PAGE and stain with coomassieblue. Cut 34-43 kDa-wide band.

[0067] Extract bands with SDS sample buffer and divide each sample intotwo portions.

[0068] Run samples on second SDS gel: one portion of each sample withoutDTT and the other with DTT.

[0069] Identify putative IDRA proteins with silver staining.

[0070] FPLC MonoQ ion exchange chromatography yielded an enrichedpreparation of Ras-protein complexes. The above noted 34-43 kDa bandappeared to be the most prominent one. Complexes with higher molecularweights were enriched as well. Ras and all species of theRas-immunoreactive complexes detected in the pooled MonoQ fractionscould be specifically immunoprecipitated by biotin-pan Ras antibody.Assuming that the larger complexes may represent multiples of the 34-43kDa complexes, the Ras-immunoreactive band with the lowest molecularweight was further purified. Two consecutive gel purification steps wereused. Following the first gel prepared under non-reducing conditions, agel slice corresponding to 34-43 kDa proteins was excised from the geland the proteins were then extracted with SDS sample buffer in theabsence or in the presence of a reducing reagent (DTT). As expected,under non-reducing conditions only the 34-43 kDa Ras-immunoreactive bandwas detected by Western immunoblotting with Ras antibody and 21 kDa Raswas released from the complexes by reduction with DTT. In addition, twomajor proteins were released by DTT from the 34-43 kDaRas-immunoreactive complexes. One was a 14-15 kDa protein and the othera 19-20 kDa protein. As the sum of the apparent molecular weights of the21 kDa H-Ras (12V) protein and each of these proteins corresponded wellto the 3443 kDa complexes, both proteins seemed like reasonablecandidates for Ras-interacting anchor. To further demonstrate that theseproteins were good candidates for molecules that specifically interactwith H-Ras (12V), the above described purification procedures wererepeated, using in parallel equal numbers of EJ cells, their parentalRat-1 cells, and myr H-Ras (12V)-transformed Rat-1 cells. The amounts ofboth the 14-15 kDa and of the 19-20 kDa proteins were significantlylower in Rat-1 cells compared to EJ cells. The 14-15 kDa protein wasbarely detected in the myr H-Ras (12V) cells. These results suggestedthat the 14-15 kDa protein interacts with the farnesylated H-Ras and maybe involved in cell transformation induced by this Ras isoform. Furtherexperiments focused on this 14-15 kDa protein.

[0071] 3. The 14-15 kDa Band was Identified as Rat Galectin-1

[0072] Quantitative and high degree of purification of the 14-15 kDarequired several gel purification steps as described in the flowdiagram. The highly purified protein released by reduction was subjectedto trypsin cleavage followed by microbore HPLC separation of the trypticfragments and MS analysis of the isolated peptides. Fragmentationpatterns of two peptides corresponded precisely to the 14 kDa ratgalectin-1. The fact that galectin is a 14 kDa protein (the size of theisolated protein) further confirmed that the FTS-sensitiveRas-interacting protein is galectin-1, a previously identified sugar-binding protein [Perillo et al., 1998]. Antibodies raised against anN-terminal peptide of galectin-1 confirmed this conclusion. Consistentwith the early observations that the Ras inhibitor FTS inhibited theformation of the cross-linked 34-43 kDa Ras-immunoreactive band (FIG.1), the amount of galectin-1 purified from FTS-treated EJ cells usingthe procedure shown in the flow diagram was very low (FIG. 2). Inaddition, immunoprecipitation of the 34-43 kDa Ras protein complexeswith biotin-Ras antibody followed by immunoblotting with galectin-1antibody revealed that galectin-1 is indeed part of the complex and thatit is released by DTT.

[0073] To be certain that the above results were not a function of useof the cross-linking reagent on isolated membranes, control andFTS-treated cells were exposed to the cross-linker DSP. Membranes wereisolated and complexes were purified by the two-step gel purificationprocedure described above. Slices of the first non-reducing gel,corresponding to 3443 kDa, 43-67 kDa, and 67-95 kDa proteins, wereexcised from the gel and subjected to the second gel in the presence ofDTT. Each sample was then immunoblotted with both Ras and galectin-1antibody. The results show that both proteins were released fromcomplexes of all sizes. These results show that H-Ras (12V) andgalectin-1 do interact in the intact cell and that they may either formcomplexes with additional proteins and/or form multimeric complexes. Inthis respect, both Ras [Inouye et al., 2000] and galectin-1 formhomodimers [Perillo et al., 1998].

[0074] In another set of experiments, intact EJ cells were treatedeither with FTS or with its inactive analog GTS (without cross-linking)and the effects of the compounds on the amounts of membrane Ras andmembrane galectin-1 were determined. In agreement with previous results[Kloog et al., 1999] FTS, but not GTS, reduced the amount of membraneRas in the cells by 50-60% (data not shown). Similarly, FTS (but notGTS) induced a 90% reduction in the amount of membrane galectin-1 (FIG.3). Thus, the magnitude of the FTS-induced galectin-1 decrease was muchgreater than that of Ras. This result demonstrates the utility ofgalectin-1 in a bioassay for FTS and its active analogs in cell cultureand intact animals, including humans.

[0075] 4. Galectin-1 Exhibits a Significant Specificity Towards H-Ras(12V)

[0076] Experiments were conducted to determine whether all the Rasisoforms interact with galectin-1. A comparative analysis was performedon the amounts of galectin-1 in three cell types: H-Ras(12V)-transformed Rat-1 (EJ) cells, N-Ras (13V)-transformed Rat-1 cells,and K-Ras 4B (12V)-transformed NIH 3T3 cells. In all of these celllines, FTS is known to dislodge Ras from cell membranes [Kloog et al.,1999]. The results showed that all of these cells expressed galectin-1,yet EJ and the K-Ras 4B cells expressed higher amounts of galectin-1compared to the N-Ras (13V) cells. Cross-linking experiments wereperformed with each of the cell lines to determine whether Ras andgalectin-1 were released from complexes of 34-43 kDa proteins.Comparable amounts of Ras were released from complexes of all celllines. By contrast, the amounts of galectin-1 released from thecomplexes were varied. Galectin-1 was very high in complexes fromH-Ras-transformed EJ cells, significantly lower in the K-Ras 4B (12V)cells, and was barely detected in the N-Ras (13V) cells. Since all ofthe cell lines tested express galectin-1 and all over-express thecorresponding Ras isoform, these results suggest that galectin-1exhibits significant specificity toward H-Ras (12V).

[0077] The above observations suggested that some of the isoforms of Rasmay prefer other IDRAs. To examine this possibility, the abovecross-linking experiment with the cell lines containing the three Rasisoforms was repeated and the release of galectin-1 and galectin-3 fromthe 34-43 kDa band isolated from cross-linked membranes was determined.The results shown in FIG. 4 indicate that Ras is released from allmembranes. K-Ras (12V) is associated with galectin-3 and H-Ras (12V) isassociated with galectin-1 and -3 in these complexes. By contrast, N-Ras(13V) anchorage to the cell membrane does not appear to be explained byeither galectin-1 or -3. As expected, myristoylated Ras is notassociated with an anchor protein as it is attached to the membrane by adifferent mechanism. The Ras in untransformed cells (Rat 1) usesgalectin-1 and -3. These observations suggest that at least two of theten known galectins may be involved in anchoring Ras to the cellmembrane.

[0078] 5. Functional Relationships Between H-Ras (12V) and Galectin-1

[0079] cDNA encoding Rat galectin-1 was obtained by RT-PCR using EJ cellRNA as a template as described in Clerch, et al. (1988). The cDNA wasinserted into pcDNA either in the sense (pcDNA-gal-1) or anti-senseorientation. Transient transfection of the sense pcDNA-gal-1 into COS-7and 293T cells resulted in a marked increase of galectin-1 as expected,due to an increase of its mRNA. To analyze the relationships between Rasand galectin-1, experiments were performed using galectin-1 antisensecDNA. Co-transfection of antisense pcDNA-gal-1 blocked of galectin-1protein expression in COS-7 or 293T cells, thus validating theefficiency of gal-i antisense. COS-7 and 293T cells were thenco-transfected with H-Ras (12V) cDNA in pcDNA or with H-Ras (12V) cDNAplus gal-1 antisense. As shown in FIG. 5, the gal-1 antisense caused amarked reduction in the concentration of H-Ras (12V). Similarexperiments were performed with myr H-Ras (12V) and with N-Ras (13V).The results showed that gal-1 antisense had no effect on theconcentration of these Ras isoforms, which are anchored by mechanismsthat do not involve galectin-1. Thus, galectin-1 contributes ratherspecifically to the expression or the stabilization of H-Ras (12V).These findings also indicate that galectin-1 antisense had the sameeffect on H-Ras protein as FTS. This observation suggests that reductionof galectin-1 could have an anticancer effect on tumors driven byoncogenic H-Ras similar to that of FTS.

[0080] In a second set of experiments, green fluorescent protein(GFP)-labeled H-Ras (12V) was expressed alone or in combination withantisense gal-1 in COS-7 and in 293T cells. Confocal microscopy was usedfor localization of the GFP-H-Ras (12V). As in previous studies withGFP-K-Ras (12V), GFP-H-Ras (12V) localized on the cell membrane [Niv etal., 1999]. The co-transfection experiments clearly showed that thegal-1 antisense induced a strong reduction in GFP-H-Ras (12V) associatedwith cell membrane (FIG. 6). It is known from previous studies thatGFP-Ras proteins, unlike their nonfused counterparts, are not readilydegraded. Indeed, in this experiment we found that the gal-1 antisensecaused a shift in the distribution of GFP-H-Ras (12V) from the cellmembrane to cytoplasmic compartments (FIG. 6) and did not reduce theamount of GFP-H-Ras (12V) expressed by the cells. These resultsdemonstrate that galectin-1 is an important protein for stabilization ofH-Ras (12V) in a manner that permits its proper localization in the cellmembrane.

EXAMPLE 2 Ras Anchor-Solid Phase Methods

[0081] Solid Phase Assays to Screen Novel Compounds that Interact withthe Ras Anchors

[0082] Two independent solid-phase methods are used to assess thepotency of new compounds as inhibitors of binding of Ras to its anchorprotein(s). In method I, Ras protein is surface-immobilized ontomicrotiter plate wells and soluble biotin-labeled Ras anchor protein(e.g., galectin-1, galectin-3, galectin-7 or galectin-8) is then boundto the immobilized Ras in the absence (100% binding) or in the presenceof various concentrations of a competitor. The apparent amount ofgalectin binding to the immobilized Ras is determined bystreptavidin-peroxidase conjugate and o-phenylenediamine as a substrate.In method II, the Ras anchor protein is surface immobilized ontomicrotiter plate wells and soluble Ras is added in the absence (100%binding) and in the presence of various concentrations of thecompetitor. Pan mouse Ras antibody, secondary biotin-goat anti mouseIgG, streptavidin-peroxidase and a substrate o-phenylenediamine areadded to determine the apparent amount of Ras binding. The reduction inOD₄₉₀ values in the presence of a competing compound as compared to theOD₄₉₀ recorded in its absence (100% binding) is indicative of the degreeof inhibition of binding.

[0083] Method I: Assay with Immobilized Ras

[0084] Fully processed HA- tagged H-Ras (12V) and HA-tagged K-Ras (12V)are produced in insect cells and purified as detailed previously (Page,M. J. et al. 1989, J. Biol. Chem. 264, 19147-19154; Lowe, P.N. 1991, J.Biol. Chem. 266, 1672-1678). Biotin-galectin-1 and biotin- galectin -3are prepared as detailed previously (Zeng, F.-J. and Gabius, H-.-J. 1993in Gabius, H.-J. and Gabius, S. eds, Lectins and gliocobiology, SpringerPub. Co. Heidelber-New York, pp. 81-85; Ander', S. et al. 1997,Bioconjugate Chem. 8, 845-855).

[0085] Mouse anti-HA antibody (1 μg/ml, Jackson ImmunoResearch)) insodium carbonate buffer pH 8.5/150 mM NaCl is added to each well for 30min, the wells are then washed with 50 mM Tris HCl buffer pH 7.4, 0.1%octylglucoside, 0.1% bovine serum albumin (BSA), 1 mM MgCl₂ (buffer A).Ras protein (0.5 μg per well) in buffer A is then added to the wells for1 h incubation at 25°. Following this Ras immobilization step and 3times wash with buffer A, biotin-galectin -1 (for H-Ras assays) orbiotin-galectin-3 (for K-Ras assays) is added in buffer A at aconcentration of 5 μg/ml in the absence or in the presence of thecompeting compound. After a 2 h-incubation period at 25°, the wells arewashed with 20 mM phosphate buffered saline pH 7.2/100 mM lactose(buffer B). The wells are then washed three times with 20 mM phosphatebuffered saline pH 7.2 (buffer C) and streptavidin-peroxidase (0.5μg/ml, Sigma) is added in buffer C for 1 h incubation at 25°. Followinga 3 times wash with buffer C, o-phenylenediamine (1 mg/ml) and H₂O₂μl/ml in buffer C are added. After 30 min-1 h incubation, the OD₄₉₀values are determined with an automated ELISA reader.

[0086] Method II. Assay with Immobilized Galectin

[0087] Galectin-1 or galectin-3 are immobilized onto microtiter plateswith asialofetuin prepared as detailed previously (Ander', S. et al.1997, Bioconjugate Chem. 8, 845-855). Basically, 1 μg asialofetuin perwell and 10 μg/ml of galectin-1 or 5 μg/ml of galectin-3 are used inbuffer C. The wells are then washed with buffer A. Ras protein (1 μg perwell in buffer A) is added in the absence and in the presence of thecompeting compound. Following a 2h-incubation period at 25°, the wellsare washed 3 times in buffer A and mouse pan Ras antibody (1 μg,Calbiochem) is added in the same buffer for a 1 h incubation at 25°.Biotin conjugated goat anti-mouse antibody (1 μg/ml, JacksonImmunoResearch) is then added, followed by streptaviden-peroxidase. Theprocedure then continues as detailed in Method I.

INDUSTRIAL APPLICABILITY

[0088] The present invention provides methods and compositionsdisrupting and inhibiting underlying biochemical reactions in variousdisease states. It also provides methods for screening compounds forpotential drugs that treat the diseases.

[0089] All patent and non-patent publications cited in thisspecification are indicative of the level of skill of those skilled inthe art to which this invention pertains. All these publications andpatent applications are herein incorporated by reference to the sameextent as if each individual publication was specifically andindividually indicated to be incorporated herein by reference.

PUBLICATIONS

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1 33 1 135 PRT Rattus sp. 1 Met Ala Cys Gly Leu Val Ala Ser Asn Leu AsnLeu Lys Pro Gly Glu 1 5 10 15 Cys Leu Lys Val Arg Gly Glu Leu Ala ProAsp Ala Lys Ser Phe Val 20 25 30 Leu Asn Leu Gly Lys Asp Ser Asn Asn LeuCys Leu His Phe Asn Pro 35 40 45 Arg Phe Asn Ala His Gly Asp Ala Asn ThrIle Val Cys Asn Ser Lys 50 55 60 Asp Asp Gly Thr Trp Gly Thr Glu Gln ArgGlu Thr Ala Phe Pro Phe 65 70 75 80 Gln Pro Gly Ser Ile Thr Glu Val CysIle Thr Phe Asp Gln Ala Asp 85 90 95 Leu Thr Ile Lys Leu Pro Asp Gly HisGlu Phe Lys Phe Pro Asn Arg 100 105 110 Leu Asn Met Glu Ala Ile Asn TyrMet Ala Ala Asp Gly Asp Phe Lys 115 120 125 Ile Lys Cys Val Ala Phe Glu130 135 2 408 DNA Rattus sp. CDS (1)..(405) 2 atg gcc tgt ggt ctg gtcgcc agc aac ctg aat ctc aaa cct ggg gaa 48 Met Ala Cys Gly Leu Val AlaSer Asn Leu Asn Leu Lys Pro Gly Glu 1 5 10 15 tgt ctc aaa gtt cgg ggagag ctg gcc ccg gac gcc aag agc ttt gtg 96 Cys Leu Lys Val Arg Gly GluLeu Ala Pro Asp Ala Lys Ser Phe Val 20 25 30 ttg aac ctg ggg aaa gac agcaac aac ctg tgc cta cac ttc aac ccc 144 Leu Asn Leu Gly Lys Asp Ser AsnAsn Leu Cys Leu His Phe Asn Pro 35 40 45 cgc ttc aac gcc cac gga gat gccaac acc att gtg tgt aac agc aag 192 Arg Phe Asn Ala His Gly Asp Ala AsnThr Ile Val Cys Asn Ser Lys 50 55 60 gac gat ggg acc tgg gga aca gaa caacgg gag act gcc ttc cct ttc 240 Asp Asp Gly Thr Trp Gly Thr Glu Gln ArgGlu Thr Ala Phe Pro Phe 65 70 75 80 cag cct ggg agc atc acg gag gtg tgcatc acc ttt gac cag gct gac 288 Gln Pro Gly Ser Ile Thr Glu Val Cys IleThr Phe Asp Gln Ala Asp 85 90 95 ctg acc atc aag ctg cca gac ggg cat gaattc aaa ttc ccc aac cgc 336 Leu Thr Ile Lys Leu Pro Asp Gly His Glu PheLys Phe Pro Asn Arg 100 105 110 ctc aac atg gag gcc atc aac tac atg gcggcg gat ggt gac ttc aag 384 Leu Asn Met Glu Ala Ile Asn Tyr Met Ala AlaAsp Gly Asp Phe Lys 115 120 125 att aag tgt gtg gcc ttt gag tga 408 IleLys Cys Val Ala Phe Glu 130 135 3 135 PRT Homo sapiens 3 Met Ala Cys GlyLeu Val Ala Ser Asn Leu Asn Leu Lys Pro Gly Glu 1 5 10 15 Cys Leu ArgVal Arg Gly Glu Val Ala Pro Asp Ala Lys Ser Phe Val 20 25 30 Leu Asn LeuGly Lys Asp Ser Asn Asn Leu Cys Leu His Phe Asn Pro 35 40 45 Arg Phe AsnAla His Gly Asp Ala Asn Thr Ile Val Cys Asn Ser Lys 50 55 60 Asp Gly GlyAla Trp Gly Thr Glu Gln Arg Glu Ala Val Phe Pro Phe 65 70 75 80 Gln ProGly Ser Val Ala Glu Val Cys Ile Thr Phe Asp Gln Ala Asn 85 90 95 Leu ThrVal Lys Leu Pro Asp Gly Tyr Glu Phe Lys Phe Pro Asn Arg 100 105 110 LeuAsn Leu Glu Ala Ile Asn Tyr Met Ala Ala Asp Gly Asp Phe Lys 115 120 125Ile Lys Cys Val Ala Phe Asp 130 135 4 408 DNA Homo sapiens CDS(1)..(405) 4 atg gct tgt ggt ctg gtc gcc agc aac ctg aat ctc aaa cct ggagag 48 Met Ala Cys Gly Leu Val Ala Ser Asn Leu Asn Leu Lys Pro Gly Glu 15 10 15 tgc ctt cga gtg cga ggc gag gtg gct cct gac gct aag agc ttc gtg96 Cys Leu Arg Val Arg Gly Glu Val Ala Pro Asp Ala Lys Ser Phe Val 20 2530 ctg aac ctg ggc aaa gac agc aac aac ctg tgc ctg cac ttc aac cct 144Leu Asn Leu Gly Lys Asp Ser Asn Asn Leu Cys Leu His Phe Asn Pro 35 40 45cgc ttc aac gcc cac ggc gac gcc aac acc atc gtg tgc aac agc aag 192 ArgPhe Asn Ala His Gly Asp Ala Asn Thr Ile Val Cys Asn Ser Lys 50 55 60 gacggc ggg gcc tgg ggg acc gag cag cgg gag gct gtc ttt ccc ttc 240 Asp GlyGly Ala Trp Gly Thr Glu Gln Arg Glu Ala Val Phe Pro Phe 65 70 75 80 cagcct gga agt gtt gca gag gtg tgc atc acc ttc gac cag gcc aac 288 Gln ProGly Ser Val Ala Glu Val Cys Ile Thr Phe Asp Gln Ala Asn 85 90 95 ctg accgtc aag ctg cca gat gga tac gaa ttc aag ttc ccc aac cgc 336 Leu Thr ValLys Leu Pro Asp Gly Tyr Glu Phe Lys Phe Pro Asn Arg 100 105 110 ctc aacctg gag gcc atc aac tac atg gca gct gac ggt gac ttc aag 384 Leu Asn LeuGlu Ala Ile Asn Tyr Met Ala Ala Asp Gly Asp Phe Lys 115 120 125 atc aaatgt gtg gcc ttt gac tga 408 Ile Lys Cys Val Ala Phe Asp 130 135 5 135PRT Mus sp. 5 Met Ala Cys Gly Leu Val Ala Ser Asn Leu Asn Leu Lys ProGly Glu 1 5 10 15 Cys Leu Lys Val Arg Gly Glu Val Ala Ser Asp Ala LysSer Phe Val 20 25 30 Leu Asn Leu Gly Lys Asp Ser Asn Asn Leu Cys Leu HisPhe Asn Pro 35 40 45 Arg Phe Asn Ala His Gly Asp Ala Asn Thr Ile Val CysAsn Thr Lys 50 55 60 Glu Asp Gly Thr Trp Gly Thr Glu His Arg Glu Pro AlaPhe Pro Phe 65 70 75 80 Gln Pro Gly Ser Ile Thr Glu Val Cys Ile Thr PheAsp Gln Ala Asp 85 90 95 Leu Thr Ile Lys Leu Pro Asp Gly His Glu Phe LysPhe Pro Asn Arg 100 105 110 Leu Asn Met Glu Ala Ile Asn Tyr Met Ala AlaAsp Gly Asp Phe Lys 115 120 125 Ile Lys Cys Val Ala Phe Glu 130 135 6250 PRT Homo sapiens 6 Met Ala Asp Asn Phe Ser Leu His Asp Ala Leu SerGly Ser Gly Asn 1 5 10 15 Pro Asn Pro Gln Gly Trp Pro Gly Ala Trp GlyAsn Gln Pro Ala Gly 20 25 30 Ala Gly Gly Tyr Pro Gly Ala Ser Tyr Pro GlyAla Tyr Pro Gly Gln 35 40 45 Ala Pro Pro Gly Ala Tyr Pro Gly Gln Ala ProPro Gly Ala Tyr Pro 50 55 60 Gly Ala Pro Gly Ala Tyr Pro Gly Ala Pro AlaPro Gly Val Tyr Pro 65 70 75 80 Gly Pro Pro Ser Gly Pro Gly Ala Tyr ProSer Ser Gly Gln Pro Ser 85 90 95 Ala Thr Gly Ala Tyr Pro Ala Thr Gly ProTyr Gly Ala Pro Ala Gly 100 105 110 Pro Leu Ile Val Pro Tyr Asn Leu ProLeu Pro Gly Gly Val Val Pro 115 120 125 Arg Met Leu Ile Thr Ile Leu GlyThr Val Lys Pro Asn Ala Asn Arg 130 135 140 Ile Ala Leu Asp Phe Gln ArgGly Asn Asp Val Ala Phe His Phe Asn 145 150 155 160 Pro Arg Phe Asn GluAsn Asn Arg Arg Val Ile Val Cys Asn Thr Lys 165 170 175 Leu Asp Asn AsnTrp Gly Arg Glu Glu Arg Gln Ser Val Phe Pro Phe 180 185 190 Glu Ser GlyLys Pro Phe Lys Ile Gln Val Leu Val Glu Pro Asp His 195 200 205 Phe LysVal Ala Val Asn Asp Ala His Leu Leu Gln Tyr Asn His Arg 210 215 220 ValLys Lys Leu Asn Glu Ile Ser Lys Leu Gly Ile Ser Gly Asp Ile 225 230 235240 Asp Leu Thr Ser Ala Ser Tyr Thr Met Ile 245 250 7 753 DNA Homosapiens CDS (1)..(750) 7 atg gca gac aat ttt tcg ctc cat gat gcg tta tctggg tct gga aac 48 Met Ala Asp Asn Phe Ser Leu His Asp Ala Leu Ser GlySer Gly Asn 1 5 10 15 cca aac cct caa gga tgg cct ggc gca tgg ggg aaccag cct gct ggg 96 Pro Asn Pro Gln Gly Trp Pro Gly Ala Trp Gly Asn GlnPro Ala Gly 20 25 30 gca ggg ggc tac cca ggg gct tcc tat cct ggg gcc tacccc ggg cag 144 Ala Gly Gly Tyr Pro Gly Ala Ser Tyr Pro Gly Ala Tyr ProGly Gln 35 40 45 gca ccc cca ggg gct tat cct gga cag gca cct cca ggc gcctac cct 192 Ala Pro Pro Gly Ala Tyr Pro Gly Gln Ala Pro Pro Gly Ala TyrPro 50 55 60 gga gca cct gga gct tat ccc gga gca cct gca cct gga gtc taccca 240 Gly Ala Pro Gly Ala Tyr Pro Gly Ala Pro Ala Pro Gly Val Tyr Pro65 70 75 80 ggg cca ccc agc ggc cct ggg gcc tac cca tct tct gga cag ccaagt 288 Gly Pro Pro Ser Gly Pro Gly Ala Tyr Pro Ser Ser Gly Gln Pro Ser85 90 95 gcc acc gga gcc tac cct gcc act ggc ccc tat ggc gcc cct gct ggg336 Ala Thr Gly Ala Tyr Pro Ala Thr Gly Pro Tyr Gly Ala Pro Ala Gly 100105 110 cca ctg att gtg cct tat aac ctg cct ttg cct ggg gga gtg gtg cct384 Pro Leu Ile Val Pro Tyr Asn Leu Pro Leu Pro Gly Gly Val Val Pro 115120 125 cgc atg ctg ata aca att ctg ggc acg gtg aag ccc aat gca aac aga432 Arg Met Leu Ile Thr Ile Leu Gly Thr Val Lys Pro Asn Ala Asn Arg 130135 140 att gct tta gat ttc caa aga ggg aat gat gtt gcc ttc cac ttt aac480 Ile Ala Leu Asp Phe Gln Arg Gly Asn Asp Val Ala Phe His Phe Asn 145150 155 160 cca cgc ttc aat gag aac aac agg aga gtc att gtt tgc aat acaaag 528 Pro Arg Phe Asn Glu Asn Asn Arg Arg Val Ile Val Cys Asn Thr Lys165 170 175 ctg gat aat aac tgg gga agg gaa gaa aga cag tcg gtt ttc ccattt 576 Leu Asp Asn Asn Trp Gly Arg Glu Glu Arg Gln Ser Val Phe Pro Phe180 185 190 gaa agt ggg aaa cca ttc aaa ata caa gta ctg gtt gaa cct gaccac 624 Glu Ser Gly Lys Pro Phe Lys Ile Gln Val Leu Val Glu Pro Asp His195 200 205 ttc aag gtt gca gtg aat gat gct cac ttg ttg cag tac aat catcgg 672 Phe Lys Val Ala Val Asn Asp Ala His Leu Leu Gln Tyr Asn His Arg210 215 220 gtt aaa aaa ctc aat gaa atc agc aaa ctg gga att tct ggt gacata 720 Val Lys Lys Leu Asn Glu Ile Ser Lys Leu Gly Ile Ser Gly Asp Ile225 230 235 240 gac ctc acc agt gct tca tat acc atg ata taa 753 Asp LeuThr Ser Ala Ser Tyr Thr Met Ile 245 250 8 250 PRT Homo sapiens 8 Met AlaAsp Asn Phe Ser Leu His Asp Ala Leu Ser Gly Ser Gly Asn 1 5 10 15 ProAsn Pro Gln Gly Trp Pro Gly Ala Trp Gly Asn Gln Pro Ala Gly 20 25 30 AlaGly Gly Tyr Pro Gly Ala Ser Tyr Pro Gly Ala Tyr Pro Gly Gln 35 40 45 AlaPro Pro Gly Ala Tyr Pro Gly Gln Ala Pro Pro Gly Ala Tyr Pro 50 55 60 GlyAla Pro Gly Ala Tyr Pro Gly Ala Pro Ala Pro Gly Val Tyr Pro 65 70 75 80Gly Pro Pro Ser Gly Pro Gly Ala Tyr Pro Ser Ser Gly Gln Pro Ser 85 90 95Ala Thr Gly Ala Tyr Pro Ala Thr Gly Pro Tyr Gly Ala Pro Ala Gly 100 105110 Pro Leu Ile Val Pro Tyr Asn Leu Pro Leu Pro Gly Gly Val Val Pro 115120 125 Arg Met Leu Ile Thr Ile Leu Gly Thr Val Lys Pro Asn Ala Asn Arg130 135 140 Ile Ala Leu Asp Phe Gln Arg Gly Asn Asp Val Ala Phe His PheAsn 145 150 155 160 Pro Arg Phe Asn Glu Asn Asn Arg Arg Val Ile Val CysAsn Thr Lys 165 170 175 Leu Asp Asn Asn Trp Gly Arg Glu Glu Arg Gln SerVal Phe Pro Phe 180 185 190 Glu Ser Gly Lys Pro Phe Lys Ile Gln Val LeuVal Glu Pro Asp His 195 200 205 Phe Lys Val Ala Val Asn Asp Ala His LeuLeu Gln Tyr Asn His Arg 210 215 220 Val Lys Lys Leu Asn Glu Ile Ser LysLeu Gly Ile Ser Gly Asp Ile 225 230 235 240 Asp Leu Thr Ser Ala Ser TyrThr Met Ile 245 250 9 753 DNA Homo sapiens CDS (1)..(750) 9 atg gca gacaat ttt tcg ctc cat gat gcg tta tct ggg tct gga aac 48 Met Ala Asp AsnPhe Ser Leu His Asp Ala Leu Ser Gly Ser Gly Asn 1 5 10 15 cca aac cctcaa gga tgg cct ggc gca tgg ggg aac cag cct gct ggg 96 Pro Asn Pro GlnGly Trp Pro Gly Ala Trp Gly Asn Gln Pro Ala Gly 20 25 30 gca ggg ggc taccca ggg gct tcc tat cct ggg gcc tac ccc ggg cag 144 Ala Gly Gly Tyr ProGly Ala Ser Tyr Pro Gly Ala Tyr Pro Gly Gln 35 40 45 gca ccc cca ggg gcttat cct gga cag gca cct cca ggc gcc tac cct 192 Ala Pro Pro Gly Ala TyrPro Gly Gln Ala Pro Pro Gly Ala Tyr Pro 50 55 60 gga gca cct gga gct tatccc gga gca cct gca cct gga gtc tac cca 240 Gly Ala Pro Gly Ala Tyr ProGly Ala Pro Ala Pro Gly Val Tyr Pro 65 70 75 80 ggg cca ccc agc ggc cctggg gcc tac cca tct tct gga cag cca agt 288 Gly Pro Pro Ser Gly Pro GlyAla Tyr Pro Ser Ser Gly Gln Pro Ser 85 90 95 gcc acc gga gcc tac cct gccact ggc ccc tat ggc gcc cct gct ggg 336 Ala Thr Gly Ala Tyr Pro Ala ThrGly Pro Tyr Gly Ala Pro Ala Gly 100 105 110 cca ctg att gtg cct tat aacctg cct ttg cct ggg gga gtg gtg cct 384 Pro Leu Ile Val Pro Tyr Asn LeuPro Leu Pro Gly Gly Val Val Pro 115 120 125 cgc atg ctg ata aca att ctgggc acg gtg aag ccc aat gca aac aga 432 Arg Met Leu Ile Thr Ile Leu GlyThr Val Lys Pro Asn Ala Asn Arg 130 135 140 att gct tta gat ttc caa agaggg aat gat gtt gcc ttc cac ttt aac 480 Ile Ala Leu Asp Phe Gln Arg GlyAsn Asp Val Ala Phe His Phe Asn 145 150 155 160 cca cgc ttc aat gag aacaac agg aga gtc att gtt tgc aat aca aag 528 Pro Arg Phe Asn Glu Asn AsnArg Arg Val Ile Val Cys Asn Thr Lys 165 170 175 ctg gat aat aac tgg ggaagg gaa gaa aga cag tcg gtt ttc cca ttt 576 Leu Asp Asn Asn Trp Gly ArgGlu Glu Arg Gln Ser Val Phe Pro Phe 180 185 190 gaa agt ggg aaa cca ttcaaa ata caa gta ctg gtt gaa cct gac cac 624 Glu Ser Gly Lys Pro Phe LysIle Gln Val Leu Val Glu Pro Asp His 195 200 205 ttc aag gtt gca gtg aatgat gct cac ttg ttg cag tac aat cat cgg 672 Phe Lys Val Ala Val Asn AspAla His Leu Leu Gln Tyr Asn His Arg 210 215 220 gtt aaa aaa ctc aat gaaatc agc aaa ctg gga att tct ggt gac ata 720 Val Lys Lys Leu Asn Glu IleSer Lys Leu Gly Ile Ser Gly Asp Ile 225 230 235 240 gac ctc acc agt gcttca tat acc atg ata taa 753 Asp Leu Thr Ser Ala Ser Tyr Thr Met Ile 245250 10 250 PRT Homo sapiens 10 Met Ala Asp Asn Phe Ser Leu His Asp AlaLeu Ser Gly Ser Gly Asn 1 5 10 15 Pro Asn Pro Gln Gly Trp Pro Gly AlaTrp Gly Asn Gln Pro Ala Gly 20 25 30 Ala Gly Gly Tyr Pro Gly Ala Ser TyrPro Gly Ala Tyr Pro Gly Gln 35 40 45 Ala Pro Pro Gly Ala Tyr Pro Gly GlnAla Pro Pro Gly Ala Tyr Pro 50 55 60 Gly Ala Pro Gly Ala Tyr Pro Gly AlaPro Ala Pro Gly Val Tyr Pro 65 70 75 80 Gly Pro Pro Ser Gly Pro Gly AlaTyr Pro Ser Ser Gly Gln Pro Ser 85 90 95 Ala Pro Gly Ala Tyr Pro Ala ThrGly Pro Tyr Gly Ala Pro Ala Gly 100 105 110 Pro Leu Ile Val Pro Tyr AsnLeu Pro Leu Pro Gly Gly Val Val Pro 115 120 125 Arg Met Leu Ile Thr IleLeu Gly Thr Val Lys Pro Asn Ala Asn Arg 130 135 140 Ile Ala Leu Asp PheGln Arg Gly Asn Asp Val Ala Phe His Phe Asn 145 150 155 160 Pro Arg PheAsn Glu Asn Asn Arg Arg Val Ile Val Cys Asn Thr Lys 165 170 175 Leu AspAsn Asn Trp Gly Arg Glu Glu Arg Gln Ser Val Phe Pro Phe 180 185 190 GluSer Gly Lys Pro Phe Lys Ile Gln Val Leu Val Glu Pro Asp His 195 200 205Phe Lys Val Ala Val Asn Asp Ala His Leu Leu Gln Tyr Asn His Arg 210 215220 Val Lys Lys Leu Asn Glu Ile Ser Lys Leu Gly Ile Ser Gly Asp Ile 225230 235 240 Asp Leu Thr Ser Ala Ser Tyr Thr Met Ile 245 250 11 250 PRTHomo sapiens 11 Met Ala Asp Asn Phe Ser Leu His Asp Ala Leu Ser Gly SerGly Asn 1 5 10 15 Pro Asn Pro Gln Gly Trp Pro Gly Ala Trp Gly Asn GlnPro Ala Gly 20 25 30 Ala Gly Gly Tyr Pro Gly Ala Ser Tyr Pro Gly Ala TyrPro Gly Gln 35 40 45 Ala Pro Pro Gly Ala Tyr Pro Gly Gln Ala Pro Pro GlyAla Tyr His 50 55 60 Gly Ala Pro Gly Ala Tyr Pro Gly Ala Pro Ala Pro GlyVal Tyr Pro 65 70 75 80 Gly Pro Pro Ser Gly Pro Gly Ala Tyr Pro Ser SerGly Gln Pro Ser 85 90 95 Ala Pro Gly Ala Tyr Pro Ala Thr Gly Pro Tyr GlyAla Pro Ala Gly 100 105 110 Pro Leu Ile Val Pro Tyr Asn Leu Pro Leu ProGly Gly Val Val Pro 115 120 125 Arg Met Leu Ile Thr Ile Leu Gly Thr ValLys Pro Asn Ala Asn Arg 130 135 140 Ile Ala Leu Asp Phe Gln Arg Gly AsnAsp Val Ala Phe His Phe Asn 145 150 155 160 Pro Arg Phe Asn Glu Asn AsnArg Arg Val Ile Val Cys Asn Thr Lys 165 170 175 Leu Asp Asn Asn Trp GlyArg Glu Glu Arg Gln Ser Val Phe Pro Phe 180 185 190 Glu Ser Gly Lys ProPhe Lys Ile Gln Val Leu Val Glu Pro Asp His 195 200 205 Phe Lys Val AlaVal Asn Asp Ala His Leu Leu Gln Tyr Asn His Arg 210 215 220 Val Lys LysLeu Asn Glu Ile Ser Lys Leu Gly Ile Ser Gly Asp Ile 225 230 235 240 AspLeu Thr Ser Ala Ser Tyr Thr Met Ile 245 250 12 250 PRT Homo sapiens 12Met Ala Asp Asn Phe Ser Leu His Asp Ala Leu Ser Gly Ser Gly Asn 1 5 1015 Pro Asn Pro Gln Gly Trp Pro Gly Ala Trp Gly Asn Gln Pro Ala Gly 20 2530 Ala Gly Gly Tyr Pro Gly Ala Ser Tyr Pro Gly Ala Tyr Pro Gly Gln 35 4045 Ala Pro Pro Gly Ala Tyr Pro Gly Gln Ala Pro Pro Gly Ala Tyr Pro 50 5560 Gly Ala Pro Gly Ala Tyr Pro Gly Ala Pro Ala Pro Gly Val Tyr Pro 65 7075 80 Gly Pro Pro Ser Gly Pro Gly Ala Tyr Pro Ser Ser Gly Gln Pro Ser 8590 95 Ala Thr Gly Ala Tyr Pro Ala Thr Gly Pro Tyr Gly Ala Pro Ala Gly100 105 110 Pro Leu Ile Val Pro Tyr Asn Leu Pro Leu Pro Gly Gly Val ValPro 115 120 125 Arg Met Leu Ile Thr Ile Leu Gly Thr Val Lys Pro Asn AlaAsn Arg 130 135 140 Ile Ala Leu Asp Phe Gln Arg Gly Asn Asp Val Ala PheHis Phe Asn 145 150 155 160 Pro Arg Phe Asn Glu Asn Asn Arg Arg Val IleVal Cys Asn Thr Lys 165 170 175 Leu Asp Asn Asn Trp Gly Arg Glu Glu ArgGln Ser Val Phe Pro Phe 180 185 190 Glu Ser Gly Lys Pro Phe Lys Ile GlnVal Leu Val Glu Pro Asp His 195 200 205 Phe Lys Val Ala Val Asn Asp AlaHis Leu Leu Gln Tyr Asn His Arg 210 215 220 Val Lys Lys Leu Asn Glu IleSer Lys Leu Gly Ile Ser Gly Asp Ile 225 230 235 240 Asp Leu Thr Ser AlaSer Tyr Thr Met Ile 245 250 13 262 PRT Rattus sp. 13 Met Ala Asp Gly PheSer Leu Asn Asp Ala Leu Ala Gly Ser Gly Asn 1 5 10 15 Pro Asn Pro GlnGly Trp Pro Gly Ala Trp Gly Asn Gln Pro Gly Ala 20 25 30 Gly Gly Tyr ProGly Ala Ser Tyr Pro Gly Ala Tyr Pro Gly Gln Ala 35 40 45 Pro Pro Gly GlyTyr Pro Gly Gln Ala Pro Pro Ser Ala Tyr Pro Gly 50 55 60 Pro Thr Gly ProSer Ala Tyr Pro Gly Pro Thr Ala Pro Gly Ala Tyr 65 70 75 80 Pro Gly ProThr Ala Pro Gly Ala Phe Pro Gly Gln Pro Gly Gly Pro 85 90 95 Gly Ala TyrPro Ser Ala Pro Gly Ala Tyr Pro Ser Ala Pro Gly Ala 100 105 110 Tyr ProAla Thr Gly Pro Phe Gly Ala Pro Thr Gly Pro Leu Thr Val 115 120 125 ProTyr Asp Met Pro Leu Pro Gly Gly Val Met Pro Arg Met Leu Ile 130 135 140Thr Ile Ile Gly Thr Val Lys Pro Asn Ala Asn Ser Ile Thr Leu Asn 145 150155 160 Phe Lys Lys Gly Asn Asp Ile Ala Phe His Phe Asn Pro Arg Phe Asn165 170 175 Glu Asn Asn Arg Arg Val Ile Val Cys Asn Thr Lys Gln Asp AsnAsn 180 185 190 Trp Gly Arg Glu Glu Arg Gln Ser Ala Phe Pro Phe Glu SerGly Lys 195 200 205 Pro Phe Lys Ile Gln Val Leu Val Glu Ala Asp His PheLys Val Ala 210 215 220 Val Asn Asp Val His Leu Leu Gln Tyr Asn His ArgMet Lys Asn Leu 225 230 235 240 Arg Glu Ile Ser Gln Leu Gly Ile Ile GlyAsp Ile Thr Leu Thr Ser 245 250 255 Ala Ser His Ala Met Ile 260 14 263PRT Mus sp. 14 Met Ala Asp Ser Phe Ser Leu Asn Asp Ala Leu Ala Gly SerGly Asn 1 5 10 15 Pro Asn Pro Gln Gly Tyr Pro Gly Ala Trp Gly Asn GlnPro Gly Ala 20 25 30 Gly Gly Tyr Pro Gly Ala Ala Tyr Pro Gly Ala Tyr ProGly Gln Ala 35 40 45 Pro Pro Gly Ala Tyr Pro Gly Gln Ala Pro Pro Gly AlaTyr Pro Gly 50 55 60 Gln Ala Pro Pro Ser Ala Tyr Pro Gly Pro Thr Ala ProGly Ala Tyr 65 70 75 80 Pro Gly Pro Thr Ala Pro Gly Ala Tyr Pro Gly GlnPro Ala Pro Gly 85 90 95 Ala Phe Pro Gly Gln Pro Gly Ala Pro Gly Ala TyrPro Gln Cys Ser 100 105 110 Gly Gly Tyr Pro Ala Ala Gly Pro Gly Val ProAla Gly Pro Leu Thr 115 120 125 Val Pro Tyr Asp Leu Pro Leu Pro Gly GlyVal Met Pro Arg Met Leu 130 135 140 Ile Thr Ile Met Gly Thr Val Lys ProAsn Ala Asn Arg Ile Val Leu 145 150 155 160 Asp Phe Arg Arg Gly Asn AspVal Ala Phe His Phe Asn Pro Arg Phe 165 170 175 Asn Glu Asn Asn Arg ArgVal Ile Val Cys Asn Thr Lys Gln Asp Asn 180 185 190 Asn Trp Gly Lys GluGlu Arg Gln Ser Ala Phe Pro Phe Glu Ser Gly 195 200 205 Lys Pro Phe LysIle Gln Val Leu Val Glu Ala Asp His Phe Lys Val 210 215 220 Ala Val AsnAsp Ala His Leu Leu Gln Tyr Asn His Arg Met Lys Asn 225 230 235 240 LeuArg Glu Ile Ser Gln Leu Gly Ile Ser Gly Asp Ile Thr Leu Thr 245 250 255Ser Ala Asn His Ala Met Ile 260 15 136 PRT Homo sapiens 15 Met Ser AsnVal Pro His Lys Ser Ser Leu Pro Glu Gly Ile Arg Pro 1 5 10 15 Gly ThrVal Leu Arg Ile Arg Gly Leu Val Pro Pro Asn Ala Ser Arg 20 25 30 Phe HisVal Asn Leu Leu Cys Gly Glu Glu Gln Gly Ser Asp Ala Ala 35 40 45 Leu HisPhe Asn Pro Arg Leu Asp Thr Ser Glu Val Val Phe Asn Ser 50 55 60 Lys GluGln Gly Ser Trp Gly Arg Glu Glu Arg Gly Pro Gly Val Pro 65 70 75 80 PheGln Arg Gly Gln Pro Phe Glu Val Leu Ile Ile Ala Ser Asp Asp 85 90 95 GlyPhe Lys Ala Val Val Gly Asp Ala Gln Tyr His His Phe Arg His 100 105 110Arg Leu Pro Leu Ala Arg Val Arg Leu Val Glu Val Gly Gly Asp Val 115 120125 Gln Leu Asp Ser Val Arg Ile Phe 130 135 16 411 DNA Homo sapiens CDS(1)..(408) 16 atg tcc aac gtc ccc cac aag tcc tcg ctg ccc gag ggc atccgc cct 48 Met Ser Asn Val Pro His Lys Ser Ser Leu Pro Glu Gly Ile ArgPro 1 5 10 15 ggc acg gtg ctg aga att cgc ggc ttg gtt cct ccc aat gccagc agg 96 Gly Thr Val Leu Arg Ile Arg Gly Leu Val Pro Pro Asn Ala SerArg 20 25 30 ttc cat gta aac ctg ctg tgc ggg gag gag cag ggc tcc gat gccgcc 144 Phe His Val Asn Leu Leu Cys Gly Glu Glu Gln Gly Ser Asp Ala Ala35 40 45 ctg cat ttc aac ccc cgg ctg gac acg tcg gag gtg gtc ttc aac agc192 Leu His Phe Asn Pro Arg Leu Asp Thr Ser Glu Val Val Phe Asn Ser 5055 60 aag gag caa ggc tcc tgg ggc cgc gag gag cgc ggg ccg ggc gtt cct240 Lys Glu Gln Gly Ser Trp Gly Arg Glu Glu Arg Gly Pro Gly Val Pro 6570 75 80 ttc cag cgc ggg cag ccc ttc gag gtg ctc atc atc gcg tca gac gac288 Phe Gln Arg Gly Gln Pro Phe Glu Val Leu Ile Ile Ala Ser Asp Asp 8590 95 ggc ttc aag gcc gtg gtt ggg gac gcc cag tac cac cac ttc cgc cac336 Gly Phe Lys Ala Val Val Gly Asp Ala Gln Tyr His His Phe Arg His 100105 110 cgc ctg ccg ctg gcg cgc gtg cgc ctg gtg gag gtg ggc ggg gac gtg384 Arg Leu Pro Leu Ala Arg Val Arg Leu Val Glu Val Gly Gly Asp Val 115120 125 cag ctg gac tcc gtg agg atc ttc tga 411 Gln Leu Asp Ser Val ArgIle Phe 130 135 17 300 PRT Homo sapiens 17 Met Met Leu Ser Leu Asn AsnLeu Gln Asn Ile Ile Tyr Ser Pro Val 1 5 10 15 Ile Pro Tyr Val Gly ThrIle Pro Asp Gln Leu Asp Pro Gly Thr Leu 20 25 30 Ile Val Ile Cys Gly HisVal Pro Ser Asp Ala Asp Arg Phe Gln Val 35 40 45 Asp Leu Gln Asn Gly SerSer Val Lys Pro Arg Ala Asp Val Ala Phe 50 55 60 His Phe Asn Pro Arg PheLys Arg Ala Gly Cys Ile Val Cys Asn Thr 65 70 75 80 Leu Ile Asn Glu LysTrp Gly Arg Glu Glu Ile Thr Tyr Asp Thr Pro 85 90 95 Phe Lys Arg Glu LysSer Phe Glu Ile Val Ile Met Val Leu Lys Asp 100 105 110 Lys Phe Gln ValPro Lys Ser Gly Thr Pro Gln Leu Pro Ser Asn Arg 115 120 125 Gly Gly AspIle Ser Lys Ile Ala Pro Arg Thr Val Tyr Thr Lys Ser 130 135 140 Lys AspSer Thr Val Asn His Thr Leu Thr Cys Thr Lys Ile Pro Pro 145 150 155 160Thr Asn Tyr Val Ser Lys Ile Leu Pro Phe Ala Ala Arg Leu Asn Thr 165 170175 Pro Met Gly Pro Gly Gly Thr Val Val Val Lys Gly Glu Val Asn Ala 180185 190 Asn Ala Lys Ser Phe Asn Val Asp Leu Leu Ala Gly Lys Ser Lys His195 200 205 Ile Ala Leu His Leu Asn Pro Arg Leu Asn Ile Lys Ala Phe ValArg 210 215 220 Asn Ser Phe Leu Gln Glu Ser Trp Gly Glu Glu Glu Arg AsnIle Thr 225 230 235 240 Ser Phe Pro Phe Ser Pro Gly Met Tyr Phe Glu MetIle Ile Tyr Cys 245 250 255 Asp Val Arg Glu Phe Lys Val Ala Val Asn GlyVal His Ser Leu Glu 260 265 270 Tyr Lys His Arg Phe Lys Glu Leu Ser SerIle Asp Thr Leu Glu Ile 275 280 285 Asn Gly Asp Ile His Leu Leu Glu ValArg Ser Trp 290 295 300 18 903 DNA Homo sapiens CDS (1)..(900) 18 atgatg ttg tcc tta aac aac cta cag aat atc atc tat agc ccg gta 48 Met MetLeu Ser Leu Asn Asn Leu Gln Asn Ile Ile Tyr Ser Pro Val 1 5 10 15 atcccg tat gtt ggc acc att ccc gat cag ctg gat cct gga act ttg 96 Ile ProTyr Val Gly Thr Ile Pro Asp Gln Leu Asp Pro Gly Thr Leu 20 25 30 att gtgata tgt ggg cat gtt cct agt gac gca gac aga ttc cag gtg 144 Ile Val IleCys Gly His Val Pro Ser Asp Ala Asp Arg Phe Gln Val 35 40 45 gat ctg cagaat ggc agc agt gtg aaa cct cga gcc gat gtg gcc ttt 192 Asp Leu Gln AsnGly Ser Ser Val Lys Pro Arg Ala Asp Val Ala Phe 50 55 60 cat ttc aat cctcgt ttc aaa agg gcc ggc tgc att gtt tgc aat act 240 His Phe Asn Pro ArgPhe Lys Arg Ala Gly Cys Ile Val Cys Asn Thr 65 70 75 80 ttg ata aat gaaaaa tgg gga cgg gaa gag atc acc tat gac acg cct 288 Leu Ile Asn Glu LysTrp Gly Arg Glu Glu Ile Thr Tyr Asp Thr Pro 85 90 95 ttc aaa aga gaa aagtct ttt gag atc gtg att atg gtg cta aag gac 336 Phe Lys Arg Glu Lys SerPhe Glu Ile Val Ile Met Val Leu Lys Asp 100 105 110 aaa ttc cag gtt ccaaag tct ggc acg ccc cag ctt cct agt aat aga 384 Lys Phe Gln Val Pro LysSer Gly Thr Pro Gln Leu Pro Ser Asn Arg 115 120 125 gga gga gac att tctaaa atc gca ccc aga act gtc tac acc aag agc 432 Gly Gly Asp Ile Ser LysIle Ala Pro Arg Thr Val Tyr Thr Lys Ser 130 135 140 aaa gat tcg act gtcaat cac act ttg act tgc acc aaa ata cca cct 480 Lys Asp Ser Thr Val AsnHis Thr Leu Thr Cys Thr Lys Ile Pro Pro 145 150 155 160 acg aac tat gtgtcg aag atc ctg cca ttc gct gca agg ttg aac acc 528 Thr Asn Tyr Val SerLys Ile Leu Pro Phe Ala Ala Arg Leu Asn Thr 165 170 175 ccc atg ggc cctggc ggc act gtc gtc gtt aaa gga gaa gtg aat gca 576 Pro Met Gly Pro GlyGly Thr Val Val Val Lys Gly Glu Val Asn Ala 180 185 190 aat gcc aaa agcttt aat gtt gac cta cta gca gga aaa tca aag cat 624 Asn Ala Lys Ser PheAsn Val Asp Leu Leu Ala Gly Lys Ser Lys His 195 200 205 att gct cta cacttg aac cca cgc ctg aat att aaa gca ttt gta aga 672 Ile Ala Leu His LeuAsn Pro Arg Leu Asn Ile Lys Ala Phe Val Arg 210 215 220 aat tct ttt cttcag gag tcc tgg gga gaa gaa gag aga aat att acc 720 Asn Ser Phe Leu GlnGlu Ser Trp Gly Glu Glu Glu Arg Asn Ile Thr 225 230 235 240 tct ttc ccattt agt cct ggg atg tac ttt gag atg ata att tat tgt 768 Ser Phe Pro PheSer Pro Gly Met Tyr Phe Glu Met Ile Ile Tyr Cys 245 250 255 gat gtt agagaa ttc aag gtt gca gta aat ggc gta cac agc ctg gag 816 Asp Val Arg GluPhe Lys Val Ala Val Asn Gly Val His Ser Leu Glu 260 265 270 tac aaa cacaga ttt aaa gag ctc agc agt att gac acg ctg gaa att 864 Tyr Lys His ArgPhe Lys Glu Leu Ser Ser Ile Asp Thr Leu Glu Ile 275 280 285 aat gga gacatc cac tta ctg gaa gta agg agc tgg tag 903 Asn Gly Asp Ile His Leu LeuGlu Val Arg Ser Trp 290 295 300 19 408 DNA Homo sapiens 19 tcagtcaaaggccacacatt tgatcttgaa gtcaccgtca gctgccatgt agttgatggc 60 ctccaggttgaggcggttgg ggaacttgaa ttcgtatcca tctggcagct tgacggtcag 120 gttggcctggtcgaaggtga tgcacacctc tgcaacactt ccaggctgga agggaaagac 180 agcctcccgctgctcggtcc cccaggcccc gccgtccttg ctgttgcaca cgatggtgtt 240 ggcgtcgccgtgggcgttga agcgagggtt gaagtgcagg cacaggttgt tgctgtcttt 300 gcccaggttcagcacgaagc tcttagcgtc aggagccacc tcgcctcgca ctcgaaggca 360 ctctccaggtttgagattca ggttgctggc gaccagacca caagccat 408 20 25 DNA Homo sapiensantisense oligonucleotide 20 aagtcaccgt cagctgccat gtagt 25 21 23 DNAHomo sapiens antisense oligonucleotide 21 gatgcacacc tctgcaacac ttc 2322 24 DNA Homo sapiens antisense oligonucleotide 22 tcagcacgaagctcttagcg tcag 24 23 22 DNA Homo sapiens antisense oligonucleotide 23gcactcgaag gcactctcca gg 22 24 22 DNA Homo sapiens antisenseoligonucleotide 24 ggttgctggc gaccagacca ca 22 25 751 DNA Homo sapiens25 ttatatcatg gtatatgaag cactggtgag gtctatgtca ccagaaattc ccagtttgct 60gatttcattg agttttttaa cccgatgatt gtactgcaac agtgagcatc attcactgca 120accttgaagt ggtcaggttc aaccagtact tgtattttga atggtttccc actttcaaat 180gggaaaaccg actgtctttc ttcccttccc cagttattat ccagctttgt attgcaaaca 240atgactctcc tgttgttctc attgaagcgt gggttaaagt ggaaggcaac atcattccct 300ctttggaaat ctaaagcaat tctgtttgca ttgggcttca ccgtgcccag aattgttatc 360agcatgcgag gcaccactcc cccaggcaaa ggcaggttat aaggcacaat cagtggccca 420gcaggggcgc cataggggcc agtggcaggg taggctccgg gggcacttgg ctgtccagaa 480gatgggtagg ccccagggcc gctgggtggc cctgggtaga ctccaggtgc ggtgctccgg 540gataagctcc aggtgctcca tggtaggcgc ctggaggtgc ctgtccagga taagcccctg 600ggggtgcctg cccggggtag gccccaggat aggaagcccc tgggtagccc cctgccccag 660caggctggtt cccccatgcg ccaggccatc cttgagggtt tgggtttcca gacccagata 720acgcatcatg gagcgaaaaa ttgtctgcca t 751 26 22 DNA Homo sapiens antisenseoligonucleotide 26 tatatgaagc actggtgagg tc 22 27 24 DNA Homo sapiensantisense oligonucleotide 27 gaagcgtggg ttaaagtgga aggc 24 28 30 DNAHomo sapiens antisense oligonucleotide 28 ttgttatcag catgcgaggcaccactcccc 30 29 21 DNA Homo sapiens antisense oligonucleotide 29cacttggctg tccagaagat g 21 30 26 DNA Homo sapiens antisenseoligonucleotide 30 gataagctcc aggtgctcca tggtag 26 31 20 DNA Homosapiens antisense oligonucleotide 31 tccagaccca gataacgcat 20 32 19 DNAHomo sapiens antisense oligonucleotide 32 tgtgggggac gttggacat 19 33 19DNA Homo sapiens antisense oligonucleotide 33 tgtttaagga caacatcat 19

1. A method for identifying a cell membrane anchor protein that binds aRas protein, comprising: preparing a first reaction mixture comprisingthe Ras protein, cell membranes or fragments thereof, and a Rasantagonist, and a second reaction mixture comprising the Ras protein andcell membranes or fragments thereof but not the Ras antagonist; adding across-linking agent to the first and second reaction mixtures wherebycross-linked complexes between the Ras protein and other proteins areproduced; separating each of the cross-linked complexes individually;identifying a complex formed in said second reaction mixture that isdisrupted by the Ras antagonist present in said first reaction mixture;separating thus-identified complex from other complexes; and separatingthe Ras protein from the other protein in the separated complex.
 2. Themethod of claim 1 wherein the antagonist is an inhibitor of a prenylatedRas protein.
 3. The method of claim 1 wherein the antagonist is aninhibitor of a farnesylated Ras protein.
 4. The method of claim 1wherein the antagonist is S-trans, trans-farnesylthiosalicylic acid(FTS) or an analog thereof.
 5. The method of claim 4 wherein the analogis 5-fluoro-FTS, 5-chloro-FTS, 4-chloro-FTS,2-chloro-5-farnesylaminobenzoic acid, farnesyl thionicoatinic acid,S-farnesyl-methylthiosalicylic acid or 3-farnesylthio-cis-acrylic acid.6. The method of claim 1 wherein the antagonist is an inhibitor of anon-prenylated Ras protein.
 7. The method of claim 1 wherein the cellmembranes are obtained from NIH fibroblasts transformed with oncogenicK-Ras 4B (12V), H-Ras (12V) or N-Ras (13V), 518A2/N-Ras melanoma cells,607B melanoma cells, Panc-1 cells containing oncogenic K-Ras, EJ cellscontaining H-Ras (12V) or MC-MA-11 cells.
 8. The method of claim 1wherein the cross-linking agent is DSS.
 9. The method of claim 1 whereinthe cross-linking agent is DSP.
 10. A method for identifying drugcandidates that inhibit aberrant Ras activity, comprising: preparing areaction mixture containing a Ras protein, an anchor protein that bindsthe Ras protein and the drug candidate; and determining effect of thedrug candidate on interaction between the Ras protein and the anchorprotein.
 11. The method of claim 10 wherein said determining comprisesmeasuring change in extent of dimerization of the Ras protein.
 12. Themethod of claim 10 wherein said determining comprises measuring changein activation of Raf protein.
 13. The method of claim 10 wherein saiddetermining comprises measuring change in extent of binding of Rafprotein to the Ras protein.
 14. The method of claim 10 wherein saiddetermining comprises measuring change in extent of binding between theRas protein and the anchor protein.
 15. The method of claim 14 whereinthe reaction mixture further comprises a cross-linking agent.
 16. Themethod of claim 10 wherein the Ras protein is immobilized on a matrix.17. The method of claim 10 wherein the anchor protein is immobilized ona matrix.
 18. The method of claim 10 wherein the anchor protein and theRas protein are in solution.
 19. The method of claim 10 wherein theanchor protein and/or the Ras protein are detectably labeled.
 20. Themethod of claim 10 wherein anchor protein and/or the Ras protein aredetectably labeled with a fluorescent protein.
 21. The method of claim20 wherein the fluorescent protein is green fluorescent protein oryellow fluorescent protein.
 22. The method of claim 10 wherein theanchor protein comprises galectin-1.
 23. The method of claim 10 whereinthe anchor protein is galectin-3.
 24. The method of claim 10 wherein theanchor protein is galectin-7.
 25. The method of claim 10 wherein theanchor protein is galectin-8.
 26. The method of claim 10 wherein the Rasprotein and the anchor protein are provided in the form of living cells.27. The method of claim 26 wherein said determining comprises measuringloss of the Ras protein from the anchor protein.
 28. The method of claim26 wherein said determining comprises observing intracellular movementof the Ras protein or the anchor protein.
 29. A method disruptingaberrant Ras activity in vivo, comprising infusing into a patientexhibiting such aberrant Ras activity, a compound comprising anoligonucleotide molecule that binds mRNA of a Ras anchor protein andinhibits expression of the Ras anchor protein.
 30. The method of claim29 wherein the oligonucleotide binds galectin-1 mRNA.
 31. The method ofclaim 29 wherein the oligonucleotide binds galectin-3 mRNA.
 32. Themethod of claim 29 wherein the oligonucleotide binds galectin-7 mRNA.33. The method of claim 29 wherein the oligonucleotide binds galectin-8mRNA.
 34. The method of claim 29 wherein the oligonucleotide contains atleast one phosphorathioate-modified nucleotide.
 35. The method of claim29 wherein the oligonucleotide is administered to the patient via aliposome.
 36. A method of determining efficacious dosages of a Rasantagonist that disrupts Ras-anchor protein binding, comprising:contacting cells with the antagonist in vivo or in vitro; collecting thecells following said contacting; isolating cell membranes from thecollected cells; measuring decrease in anchor protein concentration perunit of cell membrane protein; and correlating the decrease with dosageof the Ras antagonist.
 37. An antisense compound that specifically bindsa nucleic acid encoding galectin-1, galectin-3, galectin-7 orgalectin-8, and which and which causes degradation of the nucleic acid.38. A composition comprising the compound of claim 37 and a carrier.